Science.gov

Sample records for acid sequence-based amplification

  1. Ligation with nucleic acid sequence-based amplification.

    PubMed

    Ong, Carmichael; Tai, Warren; Sarma, Aartik; Opal, Steven M; Artenstein, Andrew W; Tripathi, Anubhav

    2012-01-01

    This work presents a novel method for detecting nucleic acid targets using a ligation step along with an isothermal, exponential amplification step. We use an engineered ssDNA with two variable regions on the ends, allowing us to design the probe for optimal reaction kinetics and primer binding. This two-part probe is ligated by T4 DNA Ligase only when both parts bind adjacently to the target. The assay demonstrates that the expected 72-nt RNA product appears only when the synthetic target, T4 ligase, and both probe fragments are present during the ligation step. An extraneous 38-nt RNA product also appears due to linear amplification of unligated probe (P3), but its presence does not cause a false-positive result. In addition, 40 mmol/L KCl in the final amplification mix was found to be optimal. It was also found that increasing P5 in excess of P3 helped with ligation and reduced the extraneous 38-nt RNA product. The assay was also tested with a single nucleotide polymorphism target, changing one base at the ligation site. The assay was able to yield a negative signal despite only a single-base change. Finally, using P3 and P5 with longer binding sites results in increased overall sensitivity of the reaction, showing that increasing ligation efficiency can improve the assay overall. We believe that this method can be used effectively for a number of diagnostic assays.

  2. RNA internal standard synthesis by nucleic acid sequence-based amplification for competitive quantitative amplification reactions.

    PubMed

    Lo, Wan-Yu; Baeumner, Antje J

    2007-02-15

    Nucleic acid sequence-based amplification (NASBA) reactions have been demonstrated to successfully synthesize new sequences based on deletion and insertion reactions. Two RNA internal standards were synthesized for use in competitive amplification reactions in which quantitative analysis can be achieved by coamplifying the internal standard with the wild type sample. The sequences were created in two consecutive NASBA reactions using the E. coli clpB mRNA sequence as model analyte. The primer sequences of the wild type sequence were maintained, and a 20-nt-long segment inside the amplicon region was exchanged for a new segment of similar GC content and melting temperature. The new RNA sequence was thus amplifiable using the wild type primers and detectable via a new inserted sequence. In the first reaction, the forwarding primer and an additional 20-nt-long sequence was deleted and replaced by a new 20-nt-long sequence. In the second reaction, a forwarding primer containing as 5' overhang sequence the wild type primer sequence was used. The presence of pure internal standard was verified using electrochemiluminescence and RNA lateral-flow biosensor analysis. Additional sequence deletion in order to shorten the internal standard amplicons and thus generate higher detection signals was found not to be required. Finally, a competitive NASBA reaction between one internal standard and the wild type sequence was carried out proving its functionality. This new rapid construction method via NASBA provides advantages over the traditional techniques since it requires no traditional cloning procedures, no thermocyclers, and can be completed in less than 4 h.

  3. Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

    PubMed Central

    Abd El Galil, Khaled H.; El Sokkary, M. A.; Kheira, S. M.; Salazar, Andre M.; Yates, Marylynn V.; Chen, Wilfred; Mulchandani, Ashok

    2005-01-01

    A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water. PMID:16269748

  4. Analytical Performance and Clinical Utility of a Nucleic Acid Sequence-Based Amplification Assay for Detection of Cytomegalovirus Infection

    PubMed Central

    Witt, Donald J.; Kemper, M.; Stead, Andrew; Sillekens, P.; Ginocchio, Christine C.; Espy, Mark J.; Paya, Carlos V.; Smith, Thomas F.; Roeles, Frits; Caliendo, Angela M.

    2000-01-01

    A nucleic acid sequence-based amplification (NASBA) assay for qualitative detection of human cytomegalovirus (CMV) pp67 mRNA was evaluated in a multicenter study. Negative results were obtained for all specimens from 50 CMV-seronegative and 50 CMV-seropositive low-risk whole-blood donors. No interference with CMV mRNA amplification was observed in the testing of 288 specimens containing various potential interfering substances, nonspecifically reacting substances (including mRNA from other herpesviruses), and three anticoagulants. A total of 95% (50 of 51) of CMV-positive (cell culture- and antigenemia immunofluorescence [AG-IFA]-positive) clinical specimens were positive by the NASBA assay. Results from different operators over multiple testing days were consistent for each of four panel members containing different concentrations of CMV mRNA, indicating the reproducibility of the assay. The estimated 95% reliable upper detection limit of the assay was 600 mRNA copies; the lower limit of detection was less than 25 mRNA copies. The clinical utility of the assay was evaluated with longitudinally collected specimens from solid-organ transplant patients (n = 21). A total of 98% (81 of 83) of the specimens from CMV-negative patients were negative by the NASBA assay, while 90% (10 of 11) of patient specimens that were positive by cell culture or AG-IFA were positive by the NASBA assay. Positive NASBA assay results were obtained earlier than AG-IFA or cell culture results for 55% of the patients and at the same time for the remainder of the patients (45%). The overall agreement between the NASBA assay and current reference tests was 86% when active CMV infection was present. These studies indicate that the CMV pp67 mRNA NASBA assay has reproducible and sensitive performance characteristics that should enable more rapid diagnosis of CMV infection. PMID:11060058

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

    PubMed

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

    2007-06-10

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

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

    PubMed Central

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

    2017-01-01

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

  7. Comparative detection of rotavirus RNA by conventional RT-PCR, TaqMan RT-PCR and real-time nucleic acid sequence-based amplification.

    PubMed

    Mo, Qiu-Hua; Wang, Hai-Bo; Tan, Hua; Wu, Bi-Mei; Feng, Zi-Li; Wang, Qi; Lin, Ji-Can; Yang, Ze

    2015-03-01

    Rotavirus is one of the major viral pathogens leading to diarrhea. Diagnosis has been conducted by either traditional cultural, serological methods or molecular biology techniques, which include RT-PCR and nucleic acid sequence-based amplification (NASBA). However, their differences regarding accuracy and sensitivity remain unknown. In this study, an in-house conventional RT-PCR assay and more importantly, an in-house real-time NASBA (RT-NASBA) were established, and compared with a commercial TaqMan RT-PCR assay. The results showed that all of these methods were able to detect and distinguish rotavirus from other diarrhea viruses with a 100% concordance rate during the course of an evaluation on 20 clinical stool samples. However, RT-NASBA was much quicker than the other two methods. More importantly, the limit of detection of RT-NASBA could reach seven copies per reaction and was one to two logs lower than that of conventional RT-PCR and TaqMan RT-PCR. These results indicate that this in-house assay was more sensitive, and thus could be used as an efficient diagnosis tool for rotavirus. To the best of our knowledge, this is the first direct comparison among three different assays for the detection of rotavirus. These findings would provide implication for the rational selection of diagnosis tool for rotavirus.

  8. Comparison and evaluation of real-time PCR, real-time nucleic acid sequence-based amplification, conventional PCR, and serology for diagnosis of Mycoplasma pneumoniae.

    PubMed

    Templeton, Kate E; Scheltinga, Sitha A; Graffelman, A Willy; Van Schie, Jolanda M; Crielaard, Jantine W; Sillekens, Peter; Van Den Broek, Peterhans J; Goossens, Herman; Beersma, Matthias F C; Claas, Eric C J

    2003-09-01

    Mycoplasma pneumoniae is a common cause of community-acquired pneumonia and lower-respiratory-tract infections. Diagnosis has traditionally been obtained by serological diagnosis, but increasingly, molecular techniques have been applied. However, the number of studies actually comparing these assays is limited. The development of a novel duplex real-time PCR assay for detection of M. pneumoniae in the presence of an internal control real-time PCR is described. In addition, real-time nucleic acid sequence-based amplification (NASBA) on an iCycler apparatus is evaluated. These assays were compared to serology and a conventional PCR assay for 106 clinical samples from patients with lower-respiratory-tract infection. Of the 106 samples, 12 (11.3%) were positive by all the molecular methods whereas serology with acute sample and convalescent samples detected 6 (5.6%) and 9 (8.5%), respectively. Clinical symptoms of the patients with Mycoplasma-positive results were compared to those of the other patients with lower-respiratory-tract infections, and it was found that the results for mean lower age numbers as well as the presence of chills, increased erythrocyte sedimentation rate, and raised C-reactive protein levels showed significant differences. Molecular methods are superior for diagnosis of M. pneumoniae, providing more timely diagnosis. In addition, using real-time methods involves less hands-on time and affords the ability to monitor the reaction in the same tube.

  9. Development of real-time multiplex nucleic acid sequence-based amplification for detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens.

    PubMed

    Loens, K; Beck, T; Ursi, D; Overdijk, M; Sillekens, P; Goossens, H; Ieven, M

    2008-01-01

    Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

  10. Detection of novel swine origin influenza A virus (H1N1) by real-time nucleic acid sequence-based amplification.

    PubMed

    Ge, Yiyue; Cui, Lunbiao; Qi, Xian; Shan, Jun; Shan, Yunfeng; Qi, Yuhua; Wu, Bing; Wang, Hua; Shi, Zhiyang

    2010-02-01

    Rapid detection of novel swine origin influenza A virus (S-OIV) (H1N1) is crucial for timely implementation of infection control measures. In this study, a haemagglutinin (HA) gene-based real-time nucleic acid sequence-based amplification (NASBA) assay was developed for the specific detection of S-OIV (H1N1). The assay was evaluated and validated by comparing it with existing detection methods for S-OIV (H1N1). Results obtained in a 10-fold dilution series assay demonstrated the analytic sensitivity of the present assay was comparable to that of a commercial S-OIV (H1N1) real-time RT-PCR kit and higher than that of the Centers for Disease Control and Prevention (CDC) TaqMan assay. The actual detection limit of the real-time NASBA assay was approximately 50 copies per reaction. Compared with reference methods (viral culture, conventional RT-PCR, and real-time RT-PCR), the sensitivity, specificity, positive predictive value, and negative predictive value of the present assay were all 100%. Overall, the results showed that the real-time NASBA assay could be used for sensitive and specific detection of S-OIV (H1N1).

  11. 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.

    PubMed

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

    2014-01-01

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

  12. Real-time nucleic acid sequence-based amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts.

    PubMed

    Hønsvall, Birgitte K; Robertson, Lucy J

    2017-01-01

    Both Cryptosporidium parvum and Cryptosporidium hominis are often associated with cryptosporidiosis in humans, but whereas humans are the main host for C. hominis, C. parvum is zoonotic and able to infect a variety of species. The oocyst transmission stages of both species of parasites are morphologically identical and molecular techniques, usually polymerase chain reaction (PCR), are required to distinguish between oocysts detected by standard methods in environmental samples, such as water. In this study, we developed two primer sets for real-time nucleic acid sequence-based amplification (NASBA), targeting the MIC1 transcript in C. parvum (CpMIC1) and C. hominis (ChMIC1). Using these primer sets, we were not only able to detect low numbers of C. parvum and C. hominis oocysts (down to 5 oocysts in 10 μl, and down to 1 oocyst using diluted RNA samples), but also distinguish between them. One of the primer sets targeted an exon only occurring in CpMIC1, thereby providing a tool for distinguishing C. parvum from other Cryptosporidium species. Although mRNA has been suggested as a tool for assessing viability of Cryptosporidium oocysts, as it is short-lived and may have high transcription, this NASBA assay detected MIC1 mRNA in inactivated oocysts. RNA within the oocysts seems to be protected from degradation, even when the oocysts have been killed by heating or freeze-thawing. Thus, our approach detects both viable and non-viable oocysts, and RNA does not seem to be a suitable marker for assessing oocyst viability.

  13. Clinical significance of expression of human cytomegalovirus pp67 late transcript in heart, lung, and bone marrow transplant recipients as determined by nucleic acid sequence-based amplification.

    PubMed

    Gerna, G; Baldanti, F; Middeldorp, J M; Furione, M; Zavattoni, M; Lilleri, D; Revello, M G

    1999-04-01

    Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of pp67 mRNA (a late viral transcript) by nucleic acid sequence-based amplification (NASBA) in a series of 50 transplant recipients, including 26 solid-organ (11 heart and 15 lung) transplant recipients (SOTRs) and 24 bone marrow transplant recipients (BMTRs). NASBA results were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in leukocytes (leukoDNAemia). On the whole, 29 patients were NASBA positive, whereas 10 were NASBA negative, and the blood of 11 patients remained HCMV negative. NASBA detected HCMV infection before quantitation of viremia did but after quantitation of leukoDNAemia and antigenemia did. In NASBA-positive blood samples, median levels of viremia, antigenemia, and leukoDNAemia were significantly higher than the relevant levels detected in NASBA-negative HCMV-positive blood samples. By using the quantitation of leukoDNAemia as the "gold standard," the analytical sensitivity (47.3%), as well as the negative predictive value (68. 3%), of NASBA for the diagnosis of HCMV infection intermediate between that of antigenemia quantitation (analytical sensitivity, 72. 3%) and that of viremia quantitation (analytical sensitivity, 28.7%), while the specificity and the positive predictive value were high (90 to 100%). However, with respect to the clinically relevant antigenemia cutoff of >/=100 used in this study for the initiation of preemptive therapy in SOTRs with reactivated HCMV infection, the clinical sensitivity of NASBA reached 100%, with a specificity of 68. 9%. Upon the initiation of antigenemia quantitation-guided treatment, the actual median antigenemia level was 158 (range, 124 to 580) in SOTRs who had reactivated infection and who presented with NASBA positivity 3.5 +/- 2.6 days in advance and 13.5 (range, 1 to 270) in the group that included BMTRs and SOTRs who had primary

  14. Human Cytomegalovirus Immediate-Early mRNA Detection by Nucleic Acid Sequence-Based Amplification as a New Parameter for Preemptive Therapy in Bone Marrow Transplant Recipients

    PubMed Central

    Gerna, Giuseppe; Baldanti, Fausto; Lilleri, Daniele; Parea, Maurizio; Alessandrino, Emilio; Pagani, Ambrogio; Locatelli, Franco; Middeldorp, Jaap; Revello, M. Grazia

    2000-01-01

    Human cytomegalovirus (HCMV) infection was monitored retrospectively by qualitative determination of immediate-early (IE) mRNA by nucleic acid sequence-based amplification (NASBA) in a series of 51 bone marrow transplant (BMT) recipients. The qualitative results for IE mRNA obtained by NASBA were compared with those obtained by prospective quantitation of HCMV viremia and antigenemia and retrospective quantitation of DNA in blood (DNAemia) by PCR as well as by qualitative determination of late pp67 mRNA by NASBA. On the whole, of the 39 HCMV-positive patients (all asymptomatic), HCMV was detected in 14 (35.9%) by quantitation of viremia, 15 (38.5%) by detection of pp67 mRNA by NASBA, 32 (82.1%) by quantitation of DNAemia, and 33 (84.6%) by quantitation of antigenemia, while HCMV was detected in 38 (97.4%) patients by detection of IE mRNA by NASBA. In the immunocompetent host, IE mRNA was not detected by NASBA in 100 blood donors or during reactivated infections in 30 breast-feeding mothers. Likewise, NASBA did not detect IE mRNA in 56 solid-organ transplant recipients in the first 21 days after transplantation. By using NASBA for detection of IE mRNA as the reference standard for detection of HCMV infection in blood samples, the diagnostic sensitivities were 67.7% for quantitation of DNAemia, 59.0% for quantitation of antigenemia, 18.3% for detection of pp67 mRNA by NASBA, and 16.0% for quantitation of viremia. Specificities and negative and positive predictive values were >90.0, >70.0, and >80.0%, respectively, for all four assays. The mean times to first HCMV detection after bone marrow transplantation were 37.7 ± 15.4 days for detection of IE mRNA by NASBA, 39.6 ± 15.6 days for quantitation of antigenemia, 40.9 ± 15.2 days for quantitation of DNAemia, and 43.7 ± 16.3 or 43.7 ± 17.5 days for quantitation of viremia and detection of pp67 mRNA by NASBA, respectively. On the whole, 31 BMT recipients received preemptive therapy by using confirmed antigenemia

  15. Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons

    PubMed Central

    Weusten, Jos J. A. M.; Carpay, Wim M.; Oosterlaken, Tom A. M.; van Zuijlen, Martien C. A.; van de Wiel, Paul A.

    2002-01-01

    For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay. PMID:11884645

  16. 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

    PubMed Central

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

    1999-01-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively. PMID:10074556

  17. 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.

    PubMed

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

    1999-04-01

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

  18. Bioanalytical applications of isothermal nucleic acid amplification techniques.

    PubMed

    Deng, Huimin; Gao, Zhiqiang

    2015-01-01

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

  19. Isothermal Amplification of Nucleic Acids.

    PubMed

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

    2015-11-25

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

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2013-01-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically. PMID:24302831

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

    PubMed

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

    2013-10-01

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

  3. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification

    PubMed Central

    Fu, Yusi; Li, Chunmei; Lu, Sijia; Zhou, Wenxiong; Tang, Fuchou; Xie, X. Sunney; Huang, Yanyi

    2015-01-01

    Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (105) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy. PMID:26340991

  4. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification.

    PubMed

    Fu, Yusi; Li, Chunmei; Lu, Sijia; Zhou, Wenxiong; Tang, Fuchou; Xie, X Sunney; Huang, Yanyi

    2015-09-22

    Whole-genome amplification (WGA) for next-generation sequencing has seen wide applications in biology and medicine when characterization of the genome of a single cell is required. High uniformity and fidelity of WGA is needed to accurately determine genomic variations, such as copy number variations (CNVs) and single-nucleotide variations (SNVs). Prevailing WGA methods have been limited by fluctuation of the amplification yield along the genome, as well as false-positive and -negative errors for SNV identification. Here, we report emulsion WGA (eWGA) to overcome these problems. We divide single-cell genomic DNA into a large number (10(5)) of picoliter aqueous droplets in oil. Containing only a few DNA fragments, each droplet is led to reach saturation of DNA amplification before demulsification such that the differences in amplification gain among the fragments are minimized. We demonstrate the proof-of-principle of eWGA with multiple displacement amplification (MDA), a popular WGA method. This easy-to-operate approach enables simultaneous detection of CNVs and SNVs in an individual human cell, exhibiting significantly improved amplification evenness and accuracy.

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

    PubMed

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

    2016-09-15

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

  6. Amplification of trace amounts of nucleic acids

    DOEpatents

    Church, George M.; Zhang, Kun

    2008-06-17

    Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (.about.6 picogram), E. coli cell (.about.5 femtogram) or Prochlorococcus cell (.about.3 femtogram).

  7. Non-instrumented nucleic acid amplification assay

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

    2008-02-01

    We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

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

    PubMed

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

    2004-05-01

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

  9. Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays.

    PubMed

    Lambert, Amy J; Martin, Denise A; Lanciotti, Robert S

    2003-01-01

    We have developed nucleic acid sequence-based amplification (NASBA), standard reverse transcription PCR (RT-PCR), and TaqMan nucleic acid amplification assays for the rapid detection of North American eastern equine encephalitis (EEE) and western equine encephalitis (WEE) viral RNAs from samples collected in the field and clinical samples. The sensitivities of these assays have been compared to that of virus isolation. While all three types of nucleic acid amplification assays provide rapid detection of viral RNAs comparable to the isolation of viruses in Vero cells, the TaqMan assays for North American EEE and WEE viral RNAs are the most sensitive. We have shown these assays to be specific for North American EEE and WEE viral RNAs by testing geographically and temporally distinct strains of EEE and WEE viruses along with a battery of related and unrelated arthropodborne viruses. In addition, all three types of nucleic acid amplification assays have been used to detect North American EEE and WEE viral RNAs from mosquito and vertebrate tissue samples. The sensitivity, specificity, and rapidity of nucleic acid amplification demonstrate the usefulness of NASBA, standard RT-PCR, and TaqMan assays, in both research and diagnostic settings, to detect North American EEE and WEE viral RNAs.

  10. Nucleic Acid Amplification Testing in Suspected Child Sexual Abuse

    ERIC Educational Resources Information Center

    Esernio-Jenssen, Debra; Barnes, Marilyn

    2011-01-01

    The American Academy of Pediatrics recommends that site-specific cultures be obtained, when indicated, for sexually victimized children. Nucleic acid amplification testing is a highly sensitive and specific methodology for identifying sexually transmitted infections. Nucleic acid amplification tests are also less invasive than culture, and this…

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

    PubMed

    Perera, Bambarendage P U; Kim, Joomyeong

    2016-02-01

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

  12. Nucleic Acid Amplification Testing for Neisseria gonorrhoeae

    PubMed Central

    Whiley, David M.; Tapsall, John W.; Sloots, Theo P.

    2006-01-01

    Nucleic acid amplification tests (NAATs) for the detection of Neisseria gonorrhoeae became available in the early 1990s. Although offering several advantages over traditional detection methods, N. gonorrhoeae NAATs do have some limitations. These include cost, risk of carryover contamination, inhibition, and inability to provide antibiotic resistance data. In addition, there are sequence-related limitations that are unique to N. gonorrhoeae NAATs. In particular, false-positive results are a major consideration. These primarily stem from the frequent horizontal genetic exchange occurring within the Neisseria genus, leading to commensal Neisseria species acquiring N. gonorrhoeae genes. Furthermore, some N. gonorrhoeae subtypes may lack specific sequences targeted by a particular NAAT. Therefore, NAAT false-negative results because of sequence variation may occur in some gonococcal populations. Overall, the N. gonorrhoeae species continues to present a considerable challenge for molecular diagnostics. The need to evaluate N. gonorrhoeae NAATs before their use in any new patient population and to educate physicians on the limitations of these tests is emphasized in this review. PMID:16436629

  13. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.

    2002-01-01

    A method of producing a plurality of a nucleic acid array, comprising, in order, the steps of amplifying in situ nucleic acid molecules of a first randomly-patterned, immobilized nucleic acid array comprising a heterogeneous pool of nucleic acid molecules affixed to a support, transferring at least a subset of the nucleic acid molecules produced by such amplifying to a second support, and affixing the subset so transferred to the second support to form a second randomly-patterned, immobilized nucleic acid array, wherein the nucleic acid molecules of the second array occupy positions that correspond to those of the nucleic acid molecules from which they were amplified on the first array, so that the first array serves as a template to produce a plurality, is disclosed.

  14. Towards Improved Accuracy of Bordetella pertussis Nucleic Acid Amplification Tests

    PubMed Central

    2012-01-01

    In many clinical microbiology laboratories, nucleic acid amplification tests such as PCR have become the routine methods for the diagnosis of pertussis. While PCR has greatly increased the ability of laboratories to detect Bordetella pertussis infections, it has also been associated with false-positive results that can, given the tendency of B. pertussis to cause outbreaks, result in unnecessary and costly control measures. The species specificity of Bordetella gene targets and their number of copies per genome greatly impact the performance characteristics of nucleic acid amplification tests for B. pertussis. It is crucial that laboratorians recognize these characteristics, to limit false-positive test results and prevent pseudo-outbreaks. PMID:22442315

  15. Increased amplification success from forensic samples with locked nucleic acids.

    PubMed

    Ballantyne, Kaye N; van Oorschot, Roland A H; Mitchell, R John

    2011-08-01

    Inadequate sample quantities and qualities can commonly result in poor DNA amplification success rates for forensic case samples. In some instances, modifying the PCR protocol or components may assist profiling by overcoming inhibition, or reducing the threshold required for successful amplification and detection. Incorporation of locked nucleic acids (LNAs) into PCR primers has previously been shown to increase amplification success for a range of non-forensic sample types and applications. To investigate their use in a forensic context, the PCR primers for four commonly used STR loci have been redesigned to include LNA bases. The modified LNA primers provided significantly increased amplification success when compared to standard DNA primers, with both high-quality buccal samples and simulated forensic casework samples. Peak heights increased by as much as 5.75× for the singleplex amplifications. When incorporated into multiplexes, the LNA primers continued to outperform standard DNA primers, with increased ease of optimisation, and increased amplification success. The use of LNAs in PCR primers can greatly assist the profiling of a range of samples, and increase success rates from challenging forensic samples.

  16. Replica amplification of nucleic acid arrays

    DOEpatents

    Church, George M.; Mitra, Robi D.

    2010-08-31

    Disclosed are improved methods of making and using immobilized arrays of nucleic acids, particularly methods for producing replicas of such arrays. Included are methods for producing high density arrays of nucleic acids and replicas of such arrays, as well as methods for preserving the resolution of arrays through rounds of replication. Also included are methods which take advantage of the availability of replicas of arrays for increased sensitivity in detection of sequences on arrays. Improved methods of sequencing nucleic acids immobilized on arrays utilizing single copies of arrays and methods taking further advantage of the availability of replicas of arrays are disclosed. The improvements lead to higher fidelity and longer read lengths of sequences immobilized on arrays. Methods are also disclosed which improve the efficiency of multiplex PCR using arrays of immobilized nucleic acids.

  17. Nucleic acid amplification using modular branched primers

    DOEpatents

    Ulanovsky, Levy; Raja, Mugasimangalam C.

    2001-01-01

    Methods and compositions expand the options for making primers for use in amplifying nucleic acid segments. The invention eliminates the step of custom synthesis of primers for Polymerase Chain Reactions (PCR). Instead of being custom-synthesized, a primer is replaced by a combination of several oligonucleotide modules selected from a pre-synthesized library. A modular combination of just a few oligonucleotides essentially mimics the performance of a conventional, custom-made primer by matching the sequence of the priming site in the template. Each oligonucleotide module has a segment that matches one of the stretches within the priming site.

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

    PubMed

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

    2007-08-29

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2016-01-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications. PMID:26906904

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

    PubMed

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

    2015-10-20

    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.

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

    PubMed Central

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

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (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. PMID:27600235

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

    PubMed

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

    2016-03-01

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

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

    Zanoli, Laura Maria; Spoto, Giuseppe

    2012-01-01

    Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

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

    PubMed

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

    2016-06-07

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

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

    PubMed Central

    Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

    2015-01-01

    Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

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

    PubMed Central

    Selck, David A.

    2016-01-01

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

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

    PubMed

    Selck, David A; Ismagilov, Rustem F

    2016-01-01

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

  10. Relevance of nucleic acid amplification techniques for diagnosis of respiratory tract infections in the clinical laboratory.

    PubMed Central

    Ieven, M; Goossens, H

    1997-01-01

    Clinical laboratories are increasingly receiving requests to perform nucleic acid amplification tests for the detection of a wide variety of infectious agents. In this paper, the efficiency of nucleic acid amplification techniques for the diagnosis of respiratory tract infections is reviewed. In general, these techniques should be applied only for the detection of microorganisms for which available diagnostic techniques are markedly insensitive or nonexistent or when turnaround times for existing tests (e.g., viral culture) are much longer than those expected with amplification. This is the case for rhinoviruses, coronaviruses, and hantaviruses causing a pulmonary syndrome, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae, and Coxiella burnetii. For Legionella spp. and fungi, contamination originating from the environment is a limiting factor in interpretation of results, as is the difficulty in differentiating colonization and infection. Detection of these agents in urine or blood by amplification techniques remains to be evaluated. In the clinical setting, there is no need for molecular diagnostic tests for the diagnosis of Pneumocystis carinii. At present, amplification methods for Mycobacterium tuberculosis cannot replace the classical diagnostic techniques, due to their lack of sensitivity and the absence of specific internal controls for the detection of inhibitors of the reaction. Also, the results of interlaboratory comparisons are unsatisfactory. Furthermore, isolates are needed for susceptibility studies. Additional work remains to be done on sample preparation methods, comparison between different amplification methods, and analysis of results. The techniques can be useful for the rapid identification of M. tuberculosis in particular circumstances, as well as the rapid detection of most rifampin-resistant isolates. The introduction of diagnostic amplification techniques into a clinical laboratory implies a level of proficiency for

  11. A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection.

    PubMed

    Tang, Ruihua; Yang, Hui; Gong, Yan; You, MinLi; Liu, Zhi; Choi, Jane Ru; Wen, Ting; Qu, Zhiguo; Mei, Qibing; Xu, Feng

    2017-03-29

    Nucleic acid testing (NAT) has been widely used for disease diagnosis, food safety control and environmental monitoring. At present, NAT mainly involves nucleic acid extraction, amplification and detection steps that heavily rely on large equipment and skilled workers, making the test expensive, time-consuming, and thus less suitable for point-of-care (POC) applications. With advances in paper-based microfluidic technologies, various integrated paper-based devices have recently been developed for NAT, which however require off-chip reagent storage, complex operation steps and equipment-dependent nucleic acid amplification, restricting their use for POC testing. To overcome these challenges, we demonstrate a fully disposable and integrated paper-based sample-in-answer-out device for NAT by integrating nucleic acid extraction, helicase-dependent isothermal amplification and lateral flow assay detection into one paper device. This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps, which could be performed by untrained users in remote settings. The proposed device consists of a sponge-based reservoir and a paper-based valve for nucleic acid extraction, an integrated battery, a PTC ultrathin heater, temperature control switch and on-chip dried enzyme mix storage for isothermal amplification, and a lateral flow test strip for naked-eye detection. It can sensitively detect Salmonella typhimurium, as a model target, with a detection limit of as low as 10(2) CFU ml(-1) in wastewater and egg, and 10(3) CFU ml(-1) in milk and juice in about an hour. This fully disposable and integrated paper-based device has great potential for future POC applications in resource-limited settings.

  12. Fluorescence detection in Lab-on-a-chip systems using ultrafast nucleic acid amplification methods

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    Today, nucleic amplification plays a key role in modern molecular biology allowing fast and specific laboratory diagnostics testing. An ultrafast microfluidic module (allowing 30 polymeric chain reaction (PCR) cycles in 6 minutes) based on an oscillating fluid plug concept was previously developed[1]. This system allows the amplification of native genomic deoxyribonucleic acid molecules (DNA) even from whole blood samples but still lacks some functionality compared to commercial bench top systems. This work presents the actual status of the renewed and advanced system, permitting the automated optical detection of not only the fluid plug position but also fluorescence detection. The system uses light emitting diodes (LED) for illumination and a low cost CMOS web-camera for optical detection. Image data processing allows the automated process control of the overall system components. Therefore, the system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the DNA molecules. The possibility to integrate swift nucleic amplification and optical detection into complex sample-to-answer analysis platforms opens up new pathways towards fast and transportable low-cost point of care devices.

  13. Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification

    PubMed Central

    Shah, Kamal G.; Guelig, Dylan; Diesburg, Steven; Buser, Joshua; Burton, Robert; LaBarre, Paul; Richards-Kortum, Rebecca; Weigl, Bernhard

    2015-01-01

    Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters. PMID:26430883

  14. Nonenzymatic catalytic signal amplification for nucleic acid hybridization assays

    NASA Technical Reports Server (NTRS)

    Fan, Wenhong (Inventor); Cassell, Alan M. (Inventor); Han, Jie (Inventor)

    2006-01-01

    Devices, methods, and kits for amplifying the signal from hybridization reactions between nucleic acid probes and their cognate targets are presented. The devices provide partially-duplexed, immobilized probe complexes, spatially separate from and separately addressable from immobilized docking strands. Cognate target acts catalytically to transfer probe from the site of probe complex immobilization to the site of immobilized docking strand, generating a detectable signal. The methods and kits of the present invention may be used to identify the presence of cognate target in a fluid sample.

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

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

    PubMed Central

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

    2014-01-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings. PMID:25089171

  17. Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout.

    PubMed

    Kong, Janay E; Wei, Qingshan; Tseng, Derek; Zhang, Jingzi; Pan, Eric; Lewinski, Michael; Garner, Omai B; Ozcan, Aydogan; Di Carlo, Dino

    2017-03-02

    Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

  18. Diagnosis of mycobacterial infections by nucleic acid amplification: 18-month prospective study.

    PubMed Central

    Kirschner, P; Rosenau, J; Springer, B; Teschner, K; Feldmann, K; Böttger, E C

    1996-01-01

    We have investigated the use of DNA amplification by PCR for the detection of mycobacteria in clinical specimens, with the gene encoding the 16S rRNA as a target. Following generic amplification of mycobacterial nucleic acids, screening was done with genus-specific probe; this was followed by species differentiation by use of highly discriminating probes or nucleic acid sequencing. In a prospective 18-month evaluation, criteria to select specimens for PCR analysis were defined. Of a total of 8,272 specimens received, 729 samples satisfied the criteria and were subjected to DNA amplification. Clinical specimens included material from the respiratory tract (sputa and bronchial washings), aspirates, biopsies, and various body fluids (cerebrospinal, pleural, peritoneal, and gastric fluids). After resolution of discrepant results, the sensitivity of the PCR assay was 84.5%, the specificity was 99.5%, the positive predictive value was 97.6%, and the negative predictive value was 96.4%. The sensitivity and negative predictive value of culture (with a combination of broth and solid media) were 77.5 and 94.8%, respectively. In conclusion, this PCR assay provides an efficient strategy to detect and identify multiple mycobacterial species and performs well in comparison with culture. PMID:8789005

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  20. Nuclemeter: A Reaction-Diffusion Based Method for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

    PubMed Central

    Liu, Changchun; Sadik, Mohamed M.; Mauk, Michael G.; Edelstein, Paul H.; Bushman, Frederic D.; Gross, Robert; Bau, Haim H.

    2014-01-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in medical and biotechnological applications. In the case of infectious diseases, such as HIV, quantification of the pathogen-load in patient specimens is critical to assess disease progression and effectiveness of drug therapy. Typically, nucleic acid quantification requires expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low-resource settings. This paper describes a simple, low-cost, reaction-diffusion 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. The method was tested for HIV viral load monitoring and performed on par with conventional benchtop methods. The proposed method is suitable for nucleic acid quantification at point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. PMID:25477046

  1. Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

    PubMed

    Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

    2014-10-13

    Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

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

    PubMed

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

    2015-10-01

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

  3. Rapid amplification/detection of nucleic acid targets utilizing a HDA/thin film biosensor.

    PubMed

    Jenison, Robert; Jaeckel, Heidi; Klonoski, Joshua; Latorra, David; Wiens, Jacinta

    2014-08-07

    Thin film biosensors exploit a flat, optically coated silicon-based surface whereupon formation of nucleic acid hybrids are enzymatically transduced in a molecular thin film that can be detected by the unaided human eye under white light. While the limit of sensitivity for detection of nucleic acid targets is at sub-attomole levels (60 000 copies) many clinical specimens containing bacterial pathogens have much lower levels of analyte present. Herein, we describe a platform, termed HDA/thin film biosensor, which performs helicase-dependant nucleic acid amplification on a thin film biosensor surface to improve the limit of sensitivity to 10 copies of the mecA gene present in methicillin-resistant strains of Staphylococcus. As double-stranded DNA is unwound by helicase it was either bound by solution-phase DNA primers to be copied by DNA polymerase or hybridized to surface immobilized probe on the thin film biosensor surface to be detected. Herein, we show that amplification reactions on the thin film biosensor are equivalent to in standard thin wall tubes, with detection at the limit of sensitivity of the assay occurring after 30 minutes of incubation time. Further we validate the approach by detecting the presence of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) from positive blood culture aliquots with high specificity (signal/noise ratio of 105).

  4. Visual, base-specific detection of nucleic acid hybridization using polymerization-based amplification.

    PubMed

    Hansen, Ryan R; Johnson, Leah M; Bowman, Christopher N

    2009-03-15

    Polymerization-based signal amplification offers sensitive visualization of biotinylated biomolecules functionalized to glass microarrays in a manner suitable for point-of-care use. Here we report using this method for visual detection of multiplexed nucleic acid hybridizations from complex media and develop an application toward point mutation detection and single nucleotide polymorphism (SNP) typing. Primer extension reactions were employed to label selectively and universally all complementary surface DNA hybrids with photoinitiators, permitting simultaneous and dynamic photopolymerization from positive sites to 0.5-nM target concentrations. Dramatic improvements in signal ratios between complementary and mismatched hybrids enabled visual discrimination of single base differences in KRAS codon-12 biomarkers.

  5. Multiplex Nucleic Acid Amplification Test for Diagnosis of Dengue Fever, Malaria, and Leptospirosis

    PubMed Central

    Waggoner, Jesse J.; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K.; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz

    2014-01-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens. PMID:24671788

  6. Sensitive detection of nucleic acids with rolling circle amplification and surface-enhanced Raman scattering spectroscopy.

    PubMed

    Hu, Juan; Zhang, Chun-yang

    2010-11-01

    Detection of specific DNA sequences is important to molecular biology research and clinical diagnostics. To improve the sensitivity of surface-enhanced Raman scattering spectroscopy (SERS), a variety of signal amplification methods has been developed, including Raman-active-dye, polymerase chain reaction (PCR) technology, molecular beacon, SERS-active substrates, and SERS-tag. However, the combination of rolling circle amplification (RCA) with SERS for nucleic acid detection has not been reported. Herein, we describe a new approach for nucleic acid detection by the combination of RCA reaction with SERS. Because of the binding of abundance repeated sequences of RCA products with gold nanoparticle (Au NP) and Rox-modified detection probes, SERS signal is significantly amplified and the detection limit of 10.0 pM might be achieved. The sensitivity of RCA-based SERS has increased by as much as 3 orders of magnitude as compared to PCR-based SERS and is also comparable with or even exceeds that of both RCA-based electrochemical and RCA-based fluorescent methods. This RCA-based SERS might discriminate perfect matched target DNA from 1-base mismatched DNA with high selectivity. The high sensitivity and selectivity of RCA-based SERS makes it a potential tool for early diagnosis of gene-related disease and also offers a great promise for multiplexed assays with DNA microarrays.

  7. Multiplex nucleic acid amplification test for diagnosis of dengue fever, malaria, and leptospirosis.

    PubMed

    Waggoner, Jesse J; Abeynayake, Janaki; Balassiano, Ilana; Lefterova, Martina; Sahoo, Malaya K; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Gresh, Lionel; Balmaseda, Angel; Harris, Eva; Banaei, Niaz; Pinsky, Benjamin A

    2014-06-01

    Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  8. 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

    PubMed Central

    Zhang, Fan; Tetali, Surya; Wang, Xue Ping; Kaplan, Mark H.; Cromme, Frans V.; Ginocchio, Christine C.

    2000-01-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82.7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease. PMID:10790122

  9. 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.

    PubMed

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

    2000-05-01

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

  10. Intraoperative diagnosis of sentinel lymph node metastases in breast cancer treatment with one-step nucleic acid amplification assay (OSNA)

    PubMed Central

    Szychta, Paweł; Westfal, Bogusław; Maciejczyk, Rafał; Smolarz, Beata; Romanowicz, Hanna; Krawczyk, Tomasz

    2016-01-01

    Introduction The aim of the study was to evaluate the clinical usefulness of a one-step nucleic acid amplification assay (OSNA) for intraoperative detection of metastases to sentinel lymph nodes (SLNs) in comparison to examination of frozen sections, and to summarize the results of previous studies. Material and methods We enrolled 98 patients aged 58.13 ±10.74 years treated surgically for breast cancer, and 99 biopsies of SLNs were followed by analysis of 105 SLNs. The central 1 mm slice of SLN was used for examination of frozen sections, whereas 2 outer slices of SLNs were analyzed intraoperatively with OSNA. Detection of isolated tumor cells (ITC), micrometastases or macrometastases with OSNA extended surgery to axillary lymph node dissection. Congruency of results was assessed between OSNA and examination of frozen sections. Results One-step nucleic acid amplification assay detected metastases in 29/105 SLNs in surgery of 27/99 breasts, including ITC in 3/29 SLNs, micrometastases in 12/29 and macrometastases in 14/29. One-step nucleic acid amplification assay detected significantly more metastases to SLNs than examination of frozen sections (p < 0.0001). All 8 inconsistent results were positive in OSNA and negative in examination of frozen sections; ITC were identified in 2/8 SLNs and micrometastases in 6/8 SLNs. Sensitivity for OSNA was calculated as 100%, specificity as 90.47%, and κ was 79.16%. Conclusions One-step nucleic acid amplification assay analysis allows rapid and quantitative detection of mRNA CK19 with high specificity and a low rate of false positives. One-step nucleic acid amplification assay is a reliable tool for intraoperative diagnosis of whole SLNs during surgery of breast cancer. One-step nucleic acid amplification assay minimizes the need for secondary surgery and avoids delays in the adjuvant treatment. PMID:27904514

  11. 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.

    PubMed

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

    2012-08-01

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

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

    PubMed

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

    2010-11-01

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

  13. Visual detection of nucleic acids based on lateral flow biosensor and hybridization chain reaction amplification.

    PubMed

    Ying, Na; Ju, Chuanjing; Li, Zhongyi; Liu, Wensen; Wan, Jiayu

    2017-03-01

    In this study, a new lateral flow nucleic acid biosensor (LFNAB) using hybridization chain reaction (HCR) for signal amplification was developed for visual detection of nucleic acids with high sensitivity and low cost. A "sandwich-type" detection strategy was employed in our design. The sandwich system of capture probe (CP)/target DNA/reporter probe (RP)-HCR complexes was fabricated as the sensing platform. As the initiator strand, reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin, and determined whether long nicked DNA polymers were formed. The biotin-labeled double-strand DNA polymers then introduced numerous Streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the lateral flow device. The CP/target DNA/RP-HCR complexes were captured on the test zone by the specific reaction between anti-Fam monoclonal antibody (anti-Fam mAb) on the test zone and Fam of the complexes. The accumulation of AuNPs on the test zone of the biosensor enabled the visual detection of specific sequences. The detection limit of specific DNA was as low as 1.76pM, which was about 2 orders lower than that of the LFNAB without HCR amplification. And the detection limit of Salmonella was 3×10(3)cfumL(-1). In conclusion, this visual detection system, HCR-LFNAB, is suitable for non-specialist personnel and point-of-care (POC) diagnosis in low-resource settings.

  14. One-step nucleic acid amplification (OSNA): where do we go with it?

    PubMed

    Tamaki, Yasuhiro

    2017-02-01

    The one-step nucleic acid amplification (OSNA) assay was initially developed for the intraoperative assessment of sentinel lymph node metastases in breast cancer. This assay measures cytokeratin 19 (CK19) mRNA copy number and is widely used in hospitals. The results of the IBCSG 23-01, ACOSOG Z0011, and AMAROS trials demonstrated that no further axillary dissection is required for patients with sentinel lymph nodes that tested positive for cancer, which has led to a decreasing trend in the need for intraoperative assessment of lymph nodes. Here, I review studies relevant to OSNA and discuss perspectives on future applications of OSNA in cancer surgery. The studies reviewed were identified by carrying out a search on PubMed for all articles pertaining to OSNA and published prior to the end of June 2016 using the keywords "OSNA" or "one-step nucleic acid amplification" in the title or abstract. Method comparison studies between OSNA and pathological assessment for the detection of lymph node metastasis in breast cancer revealed that in a pooled assessment OSNA had a high specificity (94.8 %), high concordant rate (93.8 %), and a negative predictive value (97.6 %). Similar results have been found for gastric, colorectal, and lung cancers in multicenter studies. These results demonstrate that OSNA can serve as an alternative method to pathological assessment for examining lymph node metastasis. Multicenter prospective studies with a large sample size are needed to definitively reveal the superiority of OSNA over pathological assessment to predict prognosis. Technical refinements to improve the assay are essential to its further development as a new standard for testing in place of pathological examination.

  15. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.

    PubMed

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

    2010-08-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid-based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids.

  16. Clinical impact of switching conventional enzyme immunoassay with nucleic acid amplification test for suspected Clostridium difficile-associated diarrhea.

    PubMed

    Johnson, Steven W; Kanatani, Meganne; Humphries, Romney M; Uslan, Daniel Z

    2013-04-01

    The impact of a new Clostridium difficile nucleic acid amplification test (NAAT) on antibiotic utilization in patients with suspected C difficile infection was assessed. This single-center, cross-sectional study of 270 patients demonstrated that the use of NAAT decreased antibiotic expenditure by reducing prolonged empiric days of therapy in these patients.

  17. Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip.

    PubMed

    Shen, Feng; Davydova, Elena K; Du, Wenbin; Kreutz, Jason E; Piepenburg, Olaf; Ismagilov, Rustem F

    2011-05-01

    In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader

  18. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

    PubMed Central

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

    2010-01-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

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

    PubMed Central

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

    2013-01-01

    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 sec/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 105 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. PMID:24140832

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

    PubMed

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

    2014-03-15

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

  1. A colorimetric biosensor for detection of attomolar microRNA with a functional nucleic acid-based amplification machine.

    PubMed

    Li, Dandan; Cheng, Wei; Yan, Yurong; Zhang, Ye; Yin, Yibing; Ju, Huangxian; Ding, Shijia

    2016-01-01

    A functional nucleic acid-based amplification machine was designed for simple and label-free ultrasensitive colorimetric biosensing of microRNA (miRNA). The amplification machine was composed of a complex of trigger template and C-rich DNA modified molecular beacon (MB) and G-rich DNA (GDNA) as the probe, polymerase and nicking enzyme, and a dumbbell-shaped amplification template. The presence of target miRNA triggered MB mediated strand displacement to cyclically release nicking triggers, which led to a toehold initiated rolling circle amplification to produce large amounts of GDNAs. The formed GDNAs could stack with hemin to form G-quadruplex/hemin DNAzyme, a well-known horseradish peroxidase (HRP) mimic, for catalyzing a colorimetric reaction. The modified MB improved the stringent target recognition and reduced background signal. The proposed sensing strategy showed very high sensitivity and selectivity with a wide dynamic range from 10 aM to 1.0 nM, and enabled successful visual analysis of trace amount of miRNA in real sample by the naked eye. This rapid and highly efficient signal amplification strategy provided a simple and sensitive platform for miRNA detection. It would be a versatile and powerful tool for clinical molecular diagnostics.

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

    PubMed

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

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-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.

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

    PubMed Central

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

    2016-01-01

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

  4. Multiplex-microsphere-quantitative polymerase chain reaction: nucleic acid amplification and detection on microspheres.

    PubMed

    Liang, Fang; Lai, Richard; Arora, Neetika; Zhang, Kang Liang; Yeh, Che-Cheng; Barnett, Graeme R; Voigt, Paul; Corrie, Simon R; Barnard, Ross T

    2013-01-01

    We report the development of a new system to monitor the amplification of nucleic acids on microspheres. This was realized by the design of (i) a "universal" oligonucleotide "tagged" polymerase chain reaction (PCR) forward primer, (ii) a sensor sequence complementary to the universal sequence on the forward PCR primer modified with a fluorescent dye, and (iii) a universal oligonucleotide coupled to Luminex microspheres. The PCR takes place with the microspheres present in the reaction tube. With the consumption of the universal oligonucleotide tagged forward primer, the fluorescently labeled sequences can bind to the universal oligonucleotide on the microspheres. We tested the microsphere quantitative PCR system with up to three different target genes (Neisseria meningitides porA and ctrA and influenza A M gene segment) as templates in a single PCR tube. The analytical sensitivity of this quantitative PCR system was tested and compared with the TaqMan system. The multiplex-microsphere-quantitative PCR system does not require design of unique internal probes for each target and has potential for a high degree of multiplexing, greater than the limited multiplexing achievable with current real-time protocols.

  5. Use of Nucleic Acid Amplification Tests in Tuberculosis Patients in California, 2010–2013

    PubMed Central

    Peralta, Gianna; Barry, Pennan

    2016-01-01

    Background. Nucleic acid amplification tests (NAATs) have been used as a diagnostic tool for tuberculosis (TB) in the United States for many years. We sought to assess NAAT use in TB patients in California during a period of time when NAAT availability increased throughout the world. Methods. We conducted a retrospective review of surveillance data from 6051 patients with culture-confirmed pulmonary TB who were reported to the California TB registry during 2010–2013. Results. Only 2336 of 6051 (39%) TB patients had a NAAT for diagnosis before culture results. Although 90% (N = 2101) with NAAT had positive test results, 9% (N = 217) had falsely negative NAAT results, and 0.8% (N = 18) had indeterminate NAAT results. The median time from specimen collection to TB treatment initiation was shorter when NAAT was used (3 vs 14 days, P < .0001), and patients with a positive NAAT result initiated treatment earlier than patients with a falsely negative result (1 vs 11 days from NAAT report, P < .0001). We confirmed the increased sensitivity of NAAT compared with acid-fast bacilli (AFB) smear microscopy in our study population; 92 of 145 AFB smear-negative patients had positive NAATs. Median time from specimen collection to NAAT result report differed by health jurisdiction, from 1 to 11 working days. Conclusions. Increased use of NAATs in diagnosis of pulmonary TB could decrease the time-to-treatment initiation and consequently decrease transmission. However, differential use and access to NAAT may prevent full realization of NAAT benefits in California. PMID:27957506

  6. Lyophilized Visually Readable Loop-Mediated Isothermal Reverse Transcriptase Nucleic Acid Amplification Test for Detection Ebola Zaire RNA.

    PubMed

    Carter, Christoph; Akrami, Kevan; Hall, Drew; Smith, Davey; Aronoff-Spencer, Eliah

    2017-02-24

    Recent viral outbreaks highlight the need for reliable, yet broadly deployable diagnostics for detection of epidemic and emerging pathogens. In this study we designed and optimized methods to visually detect viral nucleic acid by isothermal amplification and SYBR dye intercalation. We designed and tested loop-mediated isothermal amplification (LAMP) primers and lyophilized reactions to optimize the detection of Zaire Ebola Virus (ZEBOV) and further evolved the LAMP platform to allow room-temperature storage for deployment in resource limited settings. Our results demonstrated excellent sensitivity and specificity for viral nucleic acid sequences with lower limits of detection of less than 100 copies. Moreover, lyophilized reaction mixtures retained activity for prolonged periods under dry conditions at room temperature. This approach offers a way for detection of emerging viruses in resource limited settings.

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

    PubMed

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

    2007-12-01

    Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the MagnaPure LC Isolation instrument for nucleic acid extraction. Six hundred forty samples could be examined both by cell culture and real-time PCR. Faecal specimens (n = 285), cerebrospinal fluid (n = 210), throat swabs (n = 113), biopsies (n = 1--, vesicular fluid (n = 11), and pleural fluid specimens (n = 9) were included. By culture, 26/640 (4%) samples were positive for enterovirus. By real-time PCR, the number of positive specimens was 50 (7.8%). Of the 210 cerebrospinal fluid samples, three were positive by culture and nine by real-time PCR. Seventeen and 33 of a total of 285 faecal specimens were positive by culture and real-time PCR, respectively. In case of discrepant results, the clinical symptoms were in accordance with an infection due to enteroviruses. Genotyping using the VP1 gene correlated with serotyping by neutralization. In contrast, six of the 19 specimens that could be typed both by neutralization and by sequencing using the VP4 domain yielded a different genotype, yet within the same species. Real-time PCR turned out to be suitable for the detection of enteroviruses in the daily routine setting. In comparison to rapid culture, it offers a rapid, more sensitive, and reliable assay; especially in cerebrospinal fluid, the yield of enteroviruses is much higher.

  8. CROSS-DISCIPLINARY PHYSICS AND RELATED AREAS OF SCIENCE AND TECHNOLOGY: The structural analysis of protein sequences based on the quasi-amino acids code

    NASA Astrophysics Data System (ADS)

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

    2009-01-01

    Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Genome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system (Σ, +, *) is introduced, where Σ is the set of 64 codons. According to the characteristics of (Σ, +, *), 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, ⊕, otimes) 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).

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

    PubMed

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

    2014-08-25

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

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

    PubMed

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

    2015-11-21

    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing 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 <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products 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.

  11. A Fully Integrated Paperfluidic Molecular Diagnostic Chip for the Extraction, Amplification, and Detection of Nucleic Acids from Clinical Samples

    PubMed Central

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

    2016-01-01

    Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps onto a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in under 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings. PMID:26785636

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

    PubMed Central

    Heim, Crystal B.; Gillman, Jason D.

    2016-01-01

    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. PMID:27866151

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

    PubMed

    Heim, Crystal B; Gillman, Jason D

    2017-01-05

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

  14. Mono-allelic amplification of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and -C genes: preparation and validation of ready-to-use pre-SBT mini-kits.

    PubMed

    Dormoy, A; Froelich, N; Leisenbach, R; Weschler, B; Cazenave, J-P; Tongio, M-M

    2003-09-01

    Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.

  15. An Alignment-Free Algorithm in Comparing the Similarity of Protein Sequences Based on Pseudo-Markov Transition Probabilities among Amino Acids.

    PubMed

    Li, Yushuang; Song, Tian; Yang, Jiasheng; Zhang, Yi; Yang, Jialiang

    2016-01-01

    In this paper, we have proposed a novel alignment-free method for comparing the similarity of protein sequences. We first encode a protein sequence into a 440 dimensional feature vector consisting of a 400 dimensional Pseudo-Markov transition probability vector among the 20 amino acids, a 20 dimensional content ratio vector, and a 20 dimensional position ratio vector of the amino acids in the sequence. By evaluating the Euclidean distances among the representing vectors, we compare the similarity of protein sequences. We then apply this method into the ND5 dataset consisting of the ND5 protein sequences of 9 species, and the F10 and G11 datasets representing two of the xylanases containing glycoside hydrolase families, i.e., families 10 and 11. As a result, our method achieves a correlation coefficient of 0.962 with the canonical protein sequence aligner ClustalW in the ND5 dataset, much higher than those of other 5 popular alignment-free methods. In addition, we successfully separate the xylanases sequences in the F10 family and the G11 family and illustrate that the F10 family is more heat stable than the G11 family, consistent with a few previous studies. Moreover, we prove mathematically an identity equation involving the Pseudo-Markov transition probability vector and the amino acids content ratio vector.

  16. An Alignment-Free Algorithm in Comparing the Similarity of Protein Sequences Based on Pseudo-Markov Transition Probabilities among Amino Acids

    PubMed Central

    Li, Yushuang; Yang, Jiasheng; Zhang, Yi

    2016-01-01

    In this paper, we have proposed a novel alignment-free method for comparing the similarity of protein sequences. We first encode a protein sequence into a 440 dimensional feature vector consisting of a 400 dimensional Pseudo-Markov transition probability vector among the 20 amino acids, a 20 dimensional content ratio vector, and a 20 dimensional position ratio vector of the amino acids in the sequence. By evaluating the Euclidean distances among the representing vectors, we compare the similarity of protein sequences. We then apply this method into the ND5 dataset consisting of the ND5 protein sequences of 9 species, and the F10 and G11 datasets representing two of the xylanases containing glycoside hydrolase families, i.e., families 10 and 11. As a result, our method achieves a correlation coefficient of 0.962 with the canonical protein sequence aligner ClustalW in the ND5 dataset, much higher than those of other 5 popular alignment-free methods. In addition, we successfully separate the xylanases sequences in the F10 family and the G11 family and illustrate that the F10 family is more heat stable than the G11 family, consistent with a few previous studies. Moreover, we prove mathematically an identity equation involving the Pseudo-Markov transition probability vector and the amino acids content ratio vector. PMID:27918587

  17. Systematic Evaluation of Different Nucleic Acid Amplification Assays for Cytomegalovirus Detection: Feasibility of Blood Donor Screening.

    PubMed

    Vollmer, T; Knabbe, C; Dreier, J

    2015-10-01

    Acute primary cytomegalovirus (CMV) infections, which commonly occur asymptomatically among blood donors, represent a significant risk for serious morbidity in immunocompromised patients (a major group of transfusion recipients). We implemented a routine CMV pool screening procedure for plasma for the identification of CMV DNA-positive donors, and we evaluated the sensitivities and performance of different CMV DNA amplification systems. Minipools (MPs) of samples from 18,405 individual donors (54,451 donations) were screened for CMV DNA using the RealStar CMV PCR assay (Altona Diagnostic Technologies), with a minimum detection limit of 11.14 IU/ml. DNA was extracted with a high-volume protocol (4.8 ml, Chemagic Viral 5K kit; PerkinElmer) for blood donor pool screening (MP-nucleic acid testing [NAT]) and with the Nuclisens easyMAG system (0.5 ml; bioMérieux) for individual donation (ID)-NAT. In total, six CMV DNA-positive donors (0.03%) were identified by routine CMV screening, with DNA concentrations ranging from 4.35 × 10(2) to 4.30 × 10(3) IU/ml. Five donors already showed seroconversion and detectable IgA, IgM, and/or IgG antibody titers (IgA(+)/IgM(+)/IgG(-) or IgA(+)/IgM(+)/IgG(+)), and one donor showed no CMV-specific antibodies. Comparison of three commercial assays, i.e., the RealStar CMV PCR kit, the Sentosa SA CMV quantitative PCR kit (Vela Diagnostics), and the CMV R-gene PCR kit (bioMérieux), for MP-NAT and ID-NAT showed comparably good analytical sensitivities, ranging from 10.23 to 11.14 IU/ml (MP-NAT) or from 37.66 to 57.94 IU/ml (ID-NAT). The clinical relevance of transfusion-associated CMV infections requires further investigation, and the evaluated methods present powerful basic tools providing sensitive possibilities for viral testing. The application of CMV MP-NAT facilitated the identification of one donor with a window-phase donation during acute primary CMV infection.

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

    PubMed

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

    2016-08-15

    Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (<1 parasite μL(-1)) in a label-free and real-time manner. The developed system would be of great potential for better diagnosis of malaria in low-resource settings.

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

    DOEpatents

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

    2016-08-09

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

  20. Validation of Internal Controls for Extraction and Amplification of Nucleic Acids from Enteric Viruses in Water Samples ▿ †

    PubMed Central

    Hata, Akihiko; Katayama, Hiroyuki; Kitajima, Masaaki; Visvanathan, Chettiyappan; Nol, Chea; Furumai, Hiroaki

    2011-01-01

    Inhibitors that reduce viral nucleic acid extraction efficiency and interfere with cDNA synthesis and/or polymerase activity affect the molecular detection of viruses in aquatic environments. To overcome these significant problems, we developed a methodology for assessing nucleic acid yields and DNA amplification efficiencies for environmental water samples. This involved adding particles of adenovirus type 5 and murine norovirus and newly developed primer-sharing controls, which are amplified with the same primer pairs and result in the same amplicon sizes as the targets, to these samples. We found that nucleic acid loss during the extraction process, rather than reverse transcription-PCR (RT-PCR) inhibition, more significantly attributed to underestimation of the presence of viral genomes in the environmental water samples tested in this study. Our success rate for satisfactorily amplifying viral RNAs and DNAs by RT-PCR was higher than that for obtaining adequate nucleic acid preparations. We found that inhibitory properties were greatest when we used larger sample volumes. A magnetic silica bead-based RNA extraction method effectively removed inhibitors that interfere with viral nucleic acid extraction and RT-PCR. To our knowledge, this is the first study to assess the inhibitory properties of environmental water samples by using both control virus particles and primer-sharing controls. PMID:21602369

  1. Detection of human enteric viruses in oysters by in vivo and in vitro amplification of nucleic acids.

    PubMed Central

    Chung, H; Jaykus, L A; Sobsey, M D

    1996-01-01

    This study describes the detection of enteroviruses and hepatitis A virus in 31 naturally contaminated oyster specimens by nucleic acid amplification and oligonucleotide probing. Viruses were extracted by adsorption-elution-precipitation from 50-g oyster samples harvested from an area receiving sewage effluent discharge. Ninety percent of each extract was inoculated into primate kidney cell cultures for virus isolation and infectivity assay. Viruses in the remaining 10% of oyster extract that was not inoculated into cell cultures were further purified and concentrated by a procedure involving Freon extraction, polyethylene glycol precipitation, and Pro-Cipitate precipitation. After 3 to 4 weeks of incubation, RNA was extracted from inoculated cultures that were negative for cytopathic effects (CPE). These RNA extracts and the RNA from virions purified and concentrated directly from oyster extracts were subjected to reverse transcriptase PCR (RT-PCR) with primer pairs for human enteroviruses and hepatitis A virus. The resulting amplicons were confirmed by internal oligonucleotide probe hybridization. For the portions of oyster sample extracts inoculated into cell cultures, 12 (39%) were positive for human enteroviruses by CPE and 6 (19%) were positive by RT-PCR and oligoprobing of RNA extracts from CPE-negative cell cultures. For the remaining sample portions tested by direct RT-PCR and oligoprobing after further concentration, five (about 16%) were confirmed to be positive for human enteroviruses. Hepatitis A virus was also detected in RNA extracts of two CPE-positive samples by RT-PCR and oligoprobing. Combining the data from all three methods, enteric viruses were detected in 18 of 31 (58%) samples. Detection by nucleic acid methods increased the number of positive samples by 50% over detection by CPE in cell culture. Hence, nucleic acid amplification methods increase the detection of noncytopathic human enteric viruses in oysters. PMID:8837433

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

    PubMed

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

    2016-12-15

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

  3. Relative Accuracy of Nucleic Acid Amplification Tests and Culture in Detecting Chlamydia in Asymptomatic Men

    PubMed Central

    Cheng, Hong; Macaluso, Maurizio; Vermund, Sten H.; Hook, Edward W.

    2001-01-01

    Published estimates of the sensitivity and specificity of PCR and ligase chain reaction (LCR) for detecting Chlamydia trachomatis are potentially biased because of study design limitations (confirmation of test results was limited to subjects who were PCR or LCR positive but culture negative). Relative measures of test accuracy are less prone to bias in incomplete study designs. We estimated the relative sensitivity (RSN) and relative false-positive rate (RFP) for PCR and LCR versus cell culture among 1,138 asymptomatic men and evaluated the potential bias of RSN and RFP estimates. PCR and LCR testing in urine were compared to culture of urethral specimens. Discordant results (PCR or LCR positive, but culture negative) were confirmed by using a sequence including the other DNA amplification test, direct fluorescent antibody testing, and a DNA amplification test to detect chlamydial major outer membrane protein. The RSN estimates for PCR and LCR were 1.45 (95% confidence interval [CI] = 1.3 to 1.7) and 1.49 (95% CI = 1.3 to 1.7), respectively, indicating that both methods are more sensitive than culture. Very few false-positive results were found, indicating that the specificity levels of PCR, LCR, and culture are high. The potential bias in RSN and RFP estimates were <5 and <20%, respectively. The estimation of bias is based on the most likely and probably conservative parameter settings. If the sensitivity of culture is between 60 and 65%, then the true sensitivity of PCR and LCR is between 90 and 97%. Our findings indicate that PCR and LCR are significantly more sensitive than culture, while the three tests have similar specificities. PMID:11682509

  4. High resolution MICA genotyping by sequence-based typing (SBT).

    PubMed

    Zou, Yizhou; Stastny, Peter

    2012-01-01

    We have developed a MICA typing method based on polymerase chain reaction (PCR) sequence-based typing and a computer program that determines the polymorphisms and distinguishes the GCT repeats in exon 5. One PCR amplification was performed to obtain templates of 2.2 kb, including exons 2, 3, 4, and 5 of MICA to be sequenced with two forward and two reverse primers. Overlay of nucleotide sequencing signals resulting from presence of different GCT repeats in exon 5 from two different MICA alleles can be identified by a computer program that analyses the combined signal string containing the 35 bases.

  5. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis - Lost in the Jungle of Methods, Targets, and Assays?

    PubMed

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of 'pitfalls' is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  6. Nucleic Acid Amplification Based Diagnostic of Lyme (Neuro-)borreliosis – Lost in the Jungle of Methods, Targets, and Assays?

    PubMed Central

    Nolte, Oliver

    2012-01-01

    Laboratory based diagnosis of infectious diseases usually relies on culture of the disease causing micro-organism, followed by identification and susceptibility testing. Since Borrelia burgdorferi sensu lato, the etiologic agent of Lyme disease or Lyme borreliosis, requires very specific culture conditions (e.g. specific liquid media, long term cul-ture) traditional bacteriology is often not done on a routine basis. Instead, confirmation of the clinical diagnosis needs ei-ther indirect techniques (like serology or measurement of cellular activity in the presence of antigens) or direct but culture independent techniques, like microscopy or nucleic acid amplification techniques (NAT), with polymerase chain reaction (PCR) being the most frequently applied NAT method in routine laboratories. NAT uses nucleic acids of the disease causing micro-organism as template for amplification, isolated from various sources of clinical specimens. Although the underlying principle, adoption of the enzymatic process running during DNA duplication prior to prokaryotic cell division, is comparatively easy, a couple of ‘pitfalls’ is associated with the technique itself as well as with interpretation of the results. At present, no commercial, CE-marked and sufficiently validated PCR assay is available. A number of homebrew assays have been published, which are different in terms of target (i.e. the gene targeted by the amplification primers), method (nested PCR, PCR followed by hybridization, real-time PCR) and validation criteria. Inhibitory compounds may lead to false negative results, if no appropriate internal control is included. Carry-over of amplicons, insufficient handling and workflow and/or insufficiently validated targets/primers may result in false positive results. Different targets may yield different analytical sensitivity, depending, among other factors, of the redundancy of a target gene in the genome. Per-formance characteristics (e.g. analytical sensitivity and

  7. Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infection.

    PubMed Central

    Guatelli, J C; Gingeras, T R; Richman, D D

    1989-01-01

    The enzymatic amplification of specific nucleic acid sequences in vitro has revolutionized the use of nucleic acid hybridization assays for viral detection. With this method, the copy number of a pathogen-specific sequence is increased several orders of magnitude before detection is attempted. The sensitivity and specificity of detection are thus markedly improved. Mullis and Faloona devised the first method of sequence amplification in vitro, the polymerase chain reaction (K.B. Mullis and F.A. Faloona, Methods Enzymol. 155:355-350, 1987). By this method, synthetic oligonucleotide primers direct repeated, target-specific, deoxyribonucleic acid-synthetic reactions, resulting in an exponential increase in the amount of the specific target sequence. The application of sequence amplification to viral detection was initially performed with human immunodeficiency virus type 1 and human T-cell lymphoma virus type I. In principle, however, this approach can be applied to the detection of any deoxyribonucleic or ribonucleic acid virus; the only requirement is that sufficient nucleotide sequence data exist to allow the synthesis of target-specific oligonucleotide primers. The use of target amplification in vitro will permit a variety of studies of viral pathogenesis which have not been feasible because of the low copy number of the viral nucleic acids in infected material. This approach is particularly applicable to the study of human retroviral infections, which are chronic and persistent and are characterized by low titers of virus in tissues. In addition, target amplification in vitro will facilitate the development of new methods of sequence detection, which will be useful for rapid viral diagnosis in the clinical laboratory. PMID:2650862

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

    PubMed

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

    2006-07-17

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

  9. Effect of metallic cations on the efficiency of DNA amplification. Implications for nucleic acid replication during early stages of life

    NASA Astrophysics Data System (ADS)

    Arribas, María; de Vicente, Aránzazu; Arias, Armando; Lázaro, Ester

    2005-04-01

    The process of catalysis of biochemical reactions has been essential since the first organic molecules appeared on Earth. As the complexity of the ensemble of primitive biomolecules was very low, primitive catalysts had necessarily to be very simple molecules or ions. The evolution of catalysts had to be in parallel with the evolution of the molecular species reacting. An example of this parallel evolution is nucleic acid polymerization. Synthesis of primitive short oligonucleotides could have been catalysed by metal ions either in solution or on the surface of minerals such as montmorillonite clays. Some oligonucleotides could start to function as templates for the synthesis of complementary copies and there is experimental evidence supporting the role also played by metal ions in this process. In later stages of evolution, a group of enzymatic proteins, nucleic acid polymerases, has been selected to catalyse nucleic acid replication. The presence of Mg2+ in the active centre of these enzymes suggests that evolution has preserved some of the primitive catalysts, including them as cofactors of more complex molecules. However, the reasons why Mg2+ was selected among other ions that possibly were present in primitive environments are unknown. In this paper we try to approach this question by analysing the amplification efficiency of the polymerase chain reaction of a DNA fragment in the presence of different metal ions. In some cases the conditions of the reaction have been displaced from optimum (by the presence of nucleotide imbalances and a suboptimal Mg2+concentration). The results obtained permit one to draw interesting conclusions about how some metallic cations can help replication to proceed in conditions of limited substrate availability, a circumstance that could have been frequent at prebiotic stages, when nucleic acid synthesis was dependent on the physico-chemical conditions of the environment.

  10. fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

    PubMed Central

    Huang, Guoliang; Huang, Qin; Ma, Li; Luo, Xianbo; Pang, Biao; Zhang, Zhixin; Wang, Ruliang; Zhang, Junqi; Li, Qi; Fu, Rongxin; Ye, Jiancheng

    2014-01-01

    A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium. PMID:25475544

  11. Gonorrhoea Diagnostic and Treatment Uncertainties: Risk Factors for Culture Negative Confirmation after Positive Nucleic Acid Amplification Tests.

    PubMed

    Vyth, Rebecka; Leval, Amy; Eriksson, Björn; Ericson, Eva-Lena; Marions, Lena; Hergens, Maria-Pia

    2016-01-01

    Gonorrhoea incidence has increased substantially in Stockholm during the past years. These increases have coincided with changes in testing practice from solely culture-based to nucleic acid amplification tests (NAAT). Gonorrhoea NAAT is integrated with Chlamydia trachomatis testing and due to opportunistic screening for chlamydia, testing prevalence for gonorrhoea has increased substantially in the Stockholm population. The aim of this study was to examine epidemiological risk-factors for discordant case which are NAAT positive but culture negative. These discordant cases are especially problematic as they give rise to diagnostic and treatment uncertainties with risk for subsequent sequelae. All gonorrhoea cases from Stockholm county during 2011-2012 with at least one positive N. gonorrhoea NAAT test and follow-up cultures were included (N = 874). Data were analysed using multivariate and stratified logistic regression models. Results showed that women were 4-times more likely (OR 4.9; 95% CI 2.4-6.7) than men to have discordant cultures. Individuals tested for gonorrhoea without symptoms were 2.3 times more likely (95% CI 1.5-3.5) than those with symptoms to be discordant. NAAT method and having one week or more between NAAT and culture testing were also indicative of an increased likelihood for discordance. Using NAAT should be based on proper clinical or epidemiological indications and, when positive, followed-up with a culture-based test within one week if possible. Routine gonorrhoea testing is not recommended in low prevalence populations.

  12. A collaborative study to establish the 1st WHO International Standard for human cytomegalovirus for nucleic acid amplification technology.

    PubMed

    Fryer, Jacqueline F; Heath, Alan B; Minor, Philip D

    2016-07-01

    Variability in the performance of nucleic acid amplification technology (NAT)-based assays presents a significant problem in the diagnosis and management of human cytomegalovirus (HCMV) infections. Here we describe a collaborative study to evaluate the suitability of candidate reference materials to harmonize HCMV viral load measurements in a wide range of NAT assays. Candidate materials comprised lyophilized Merlin virus, liquid Merlin virus, liquid AD169 virus, and purified HCMV Merlin DNA cloned into a bacterial artificial chromosome. Variability in the laboratory mean HCMV concentrations determined for virus samples across the different assays was 2 log10. Variability for the purified DNA sample was higher (>3 log10). The agreement between laboratories was markedly improved when the potencies of the liquid virus samples were expressed relative to the lyophilized virus candidate. In contrast, the agreement between laboratories for the purified DNA sample was not improved. Results indicated the suitability of the lyophilized Merlin virus preparation as the 1st WHO International Standard for HCMV for NAT. It was established in October 2010, with an assigned potency of 5 × 10(6) International Units (IU) (NIBSC code 09/162). It is intended to be used to calibrate secondary references, used in HCMV NAT assays, in IU.

  13. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing].

    PubMed

    Dmitrienko, E V; Khomiakova, E A; Pyshnaia; Bragin, A G; Vedernikov, V E; Pyshnyĭ, D V

    2010-01-01

    The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

  14. Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).

    PubMed

    Lutz, Sascha; Weber, Patrick; Focke, Max; Faltin, Bernd; Hoffmann, Jochen; Müller, Claas; Mark, Daniel; Roth, Günter; Munday, Peter; Armes, Niall; Piepenburg, Olaf; Zengerle, Roland; von Stetten, Felix

    2010-04-07

    For the first time we demonstrate a self-sufficient lab-on-a-foil system for the fully automated analysis of nucleic acids which is based on the recently available isothermal recombinase polymerase amplification (RPA). The system consists of a novel, foil-based centrifugal microfluidic cartridge including prestored liquid and dry reagents, and a commercially available centrifugal analyzer for incubation at 37 degrees C and real-time fluorescence detection. The system was characterized with an assay for the detection of the antibiotic resistance gene mecA of Staphylococcus aureus. The limit of detection was <10 copies and time-to-result was <20 min. Microfluidic unit operations comprise storage and release of liquid reagents, reconstitution of lyophilized reagents, aliquoting the sample into < or = 30 independent reaction cavities, and mixing of reagents with the DNA samples. The foil-based cartridge was produced by blow-molding and sealed with a self-adhesive tape. The demonstrated system excels existing PCR based lab-on-a-chip platforms in terms of energy efficiency and time-to-result. Applications are suggested in the field of mobile point-of-care analysis, B-detection, or in combination with continuous monitoring systems.

  15. Clinical implications of nucleic acid amplification methods for the diagnosis of viral infections of the nervous system.

    PubMed

    Weber, T; Frye, S; Bodemer, M; Otto, M; Lüke, W

    1996-06-01

    Amplification of viral nucleic acids from the cerebrospinal fluid (CSF) has considerably improved the diagnosis of several acute, subacute and chronic viral infections of the nervous system. In herpes simplex virus (HSV) encephalitis (HSE) the polymerase chain reaction (PCR) has become the method of choice for the rapid, non invasive diagnosis. Other herpes virus associated diseases which can now be reliably diagnosed are encephalitis, ventriculoencephalitis, polymyeloradiculitis, myelitis and an inflammatory polyradiculoneuropathy caused by cytomegalovirus (CMV), HSV, varicella-zoster virus (VZV) or Epstein-Barr virus (EBV), EBV associated primary B-cell-lymphoma of the brain, acute aseptic meningitis in young adults allied with VZV, and meningoencephalitis with recurrent seizures due to human herpes virus type 6 (HHV-6). In AIDS patients, PCR has helped to differentiate lesions either due to the human immunodeficiency virus (HIV) itself or to opportunistic infections such as progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) or CMV related complications. HIV can be detected early in the course of infection in the CSF and the amount of proviral DNA in CSF cells seems to be correlated with the severity and/or progression of neurological signs and symptoms. Acute epidemic aseptic meningitis caused by enterovirus infections can now be reliably diagnosed and typed by reverse transcriptase PCR (RT-PCR). Meningitis cases caused by vaccination with the Jeryl Lynn and Urabe vaccine strain of mumps virus have been identified using RT-PCR and sequencing of the amplified products (amplicon).

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

    PubMed Central

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

    2017-01-01

    Recently, a number of techniques have been approved for quantification of viral nucleic acids in clinical samples. Viral load (VL) tests have considerable importance in the management of patients and are widely used in routine diagnosis. In clinical virology, VL testing are important to monitor the antiviral treatment, to initiate preemptive therapy, to understand pathogenesis, and to evaluate the infectivity. These tests have now become a part of many diagnostic and treatment guidelines. Considering the various challenges for in-house viral testing related to the standardization, validation, and precision; they are gradually being replaced by the United States Food and Drug Administration (US FDA) cleared tests. This review summarizes the various viral quantification methods and also discusses the clinical applicability of these in human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, Cytomegalovirus, and Epstein Barr virus infected patients. Further the challenges and future perspectives of VL testing have also been discussed. PMID:28251100

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

    PubMed

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

    2017-01-01

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

  18. Prospective evaluation of the Alere i Influenza A&B nucleic acid amplification versus Xpert Flu/RSV.

    PubMed

    Nguyen Van, J C; Caméléna, F; Dahoun, M; Pilmis, B; Mizrahi, A; Lourtet, J; Behillil, S; Enouf, V; Le Monnier, A

    2016-05-01

    The rapid and accurate detection of influenza virus in respiratory specimens is required for optimal management of patients with acute respiratory infections. Because of the variability of the symptoms and the numerous other causes of influenza-like illness, the diagnosis of influenza cannot be made on the basis of clinical criteria alone. Thus, rapid influenza diagnostic tests have been developed such as the Alere i Influenza A&B isothermal nucleic acid assay. We prospectively evaluated the performance of the Alere i Influenza A&B assay in comparison with our routine Xpert Flu/RSV assay. Positive samples were subtyped according to the protocol from the National Influenza Center (Paris, France). A total of 96 respiratory nasal swab samples were analyzed: with both methods, 38 were positive and 56 were negative. Samples were prospectively collected from January 20 to April 8, 2015, from patient (86 adult and 10 pediatric patients) presenting with an influenza-like illness through the French influenza season. In comparison with the Xpert Flu/RSV assay, the overall sensitivity and specificity of the Alere i Influenza A&B assay were 95% and 100%, respectively. Our results indicate that the Alere i Influenza A&B assay has a good overall analytical performance and a high degree of concordance with the PCR-based Xpert Flu/RSV assay. The Alere i Influenza A&B isothermal nucleic acid amplification test is a powerful tool for influenza detection due to its high sensitivity and specificity as well as its ability to generate results within 15min.

  19. Nucleic acid-amplification testing for hepatitis B in cornea donors.

    PubMed

    Fornés, Maria Gema; Jiménez, Maria Angustias; Eisman, Marcela; Gómez Villagrán, Jose Luis; Villalba, Rafael

    2016-06-01

    Careful donor selection and implementation of tests of appropriate sensitivity and specificity are of paramount importance for minimizing the risk of transmitting infectious diseases from donors to corneal allograft recipients. Reported cases of viral transmission with corneal grafts are very unusual. Nevertheless potential virus transmission through the engraftment cannot be ruled out. According to European Guideline 2006/17/EC, screening for antibodies for Hepatitis B core antigen (anti HBc) is mandatory, and when this test is positive, some criteria must be established before using corneas. Despite the continuous progress in screening tests, donors carrying an occult hepatitis B infection (OBI) can cause transplant-transmitted hepatitis B. To date, Nucleic Acid Testing (NAT) is not an obligatory assay in corneal tissue setting neither in our country nor in the rest of European countries. Herein, we report three cornea donors that were rejected with the diagnosis of OBI through the testing of sensitive NAT and the serological profile of Hepatitis B virus. The aim of this report is to emphasize the need to include NAT in new reviews of EU Tissues and Cells Directives in order to increase level of security in tissue donation as well as not to reject a high number of donors with isolated profile of anti HBc in geographical areas with high prevalence of Hepatitis B, that could be rejected without a true criterion of Hepatitis B infection.

  20. Genomic detection of human immunodeficiency virus (HIV) by nucleic acid amplification test in a frequent platelet donor during the pre-seroconversion period.

    PubMed

    Pondé, Robério Amorim de Almeida

    2011-11-01

    Since serological donor-screening tests for HIV were introduced in 1985, the safety of donated blood components has improved dramatically. However, these tests do not completely prevent the risk of transfusion-associated HIV infection related to the use of blood donated during the pre-seroconversion window period. Testing based on nucleic acid amplification is being implemented to screen for HIV-infected blood donated during this period, which has reduced the probability of transmitting HIV through transfusion by shortening the window period. This article describes a case of acute HIV-1 infection, detected using a nucleic acid amplification test (NAT) in a repeat blood donor who donated during the pre-seroconversion window period and whose antigen and anti-HIV antibody expression was observed after molecular marker detection. In addition, the possible route of infection is discussed based on the patient's history, and finally, the need for NAT technology for blood donor screening is emphasized.

  1. Multiplex, Rapid, and Sensitive Isothermal Detection of Nucleic-Acid Sequence by Endonuclease Restriction-Mediated Real-Time Multiple Cross Displacement Amplification.

    PubMed

    Wang, Yi; Wang, Yan; Zhang, Lu; Liu, Dongxin; Luo, Lijuan; Li, Hua; Cao, Xiaolong; Liu, Kai; Xu, Jianguo; Ye, Changyun

    2016-01-01

    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 labeled 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 min, 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.

  2. Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

    PubMed

    Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

    2012-12-01

    A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.

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

    PubMed

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

    2015-01-01

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

  4. Evaluation of Six Commercial Nucleic Acid Amplification Tests for Detection of Neisseria gonorrhoeae and Other Neisseria Species▿

    PubMed Central

    Tabrizi, Sepehr N.; Unemo, Magnus; Limnios, Athena E.; Hogan, Tiffany R.; Hjelmevoll, Stig-Ove; Garland, Susanne M.; Tapsall, John

    2011-01-01

    Molecular detection of Neisseria gonorrhoeae in extragenital samples may result in false-positive results due to cross-reaction with commensal Neisseria species or Neisseria meningitidis. This study examined 450 characterized clinical culture isolates, comprising 216 N. gonorrhoeae isolates and 234 isolates of nongonococcal Neisseria species (n = 218) and 16 isolates of other closely related bacteria, with six commercial nucleic acid amplification tests (NAATs). The six NAATs tested were Gen-Probe APTIMA COMBO 2 and APTIMA GC, Roche COBAS Amplicor CT/NG and COBAS 4800 CT/NG tests, BD ProbeTec GC Qx amplified DNA assay, and Abbott RealTime CT/NG test. All assays except COBAS Amplicor CT/NG test where four (1.9%) isolates were not detected showed a positive result with all N. gonorrhoeae isolates (n = 216). Among the 234 nongonococcal isolates examined, initial results from all assays displayed some false-positive results due to cross-reactions. Specifically, the COBAS Amplicor and ProbeTec tests showed the highest number of false-positive results, detecting 33 (14.1%) and 26 (11%) nongonococcal Neisseria isolates, respectively. On the first testing, APTIMA COMBO 2, APTIMA GC, Abbott RealTime, and Roche COBAS 4800 showed lower level of cross-reactions with five (2.1%), four (1.7%), two (1%), and two (1%) of the isolates showing low-level positivity, respectively. Upon retesting of these nine nongonococcal isolates using freshly cultured colonies, none were positive by the APTIMA COMBO 2, Abbott RealTime, or COBAS 4800 test. In conclusion, the COBAS Amplicor and ProbeTec tests displayed high number of false-positive results, while the remaining NAATs showed only sporadic low-level false-positive results. Supplementary testing for confirmation of N. gonorrhoeae NAATs remains recommended with all samples tested, in particular those from extragenital sites. PMID:21813721

  5. Nucleic Acid Amplification Testing and Sequencing Combined with Acid-Fast Staining in Needle Biopsy Lung Tissues for the Diagnosis of Smear-Negative Pulmonary Tuberculosis

    PubMed Central

    Tian, Panwen; Chen, Xuerong; Liang, Zongan

    2016-01-01

    Background Smear-negative pulmonary tuberculosis (PTB) is common and difficult to diagnose. In this study, we investigated the diagnostic value of nucleic acid amplification testing and sequencing combined with acid-fast bacteria (AFB) staining of needle biopsy lung tissues for patients with suspected smear-negative PTB. Methods Patients with suspected smear-negative PTB who underwent percutaneous transthoracic needle biopsy between May 1, 2012, and June 30, 2015, were enrolled in this retrospective study. Patients with AFB in sputum smears were excluded. All lung biopsy specimens were fixed in formalin, embedded in paraffin, and subjected to acid-fast staining and tuberculous polymerase chain reaction (TB-PCR). For patients with positive AFB and negative TB-PCR results in lung tissues, probe assays and 16S rRNA sequencing were used for identification of nontuberculous mycobacteria (NTM). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and diagnostic accuracy of PCR and AFB staining were calculated separately and in combination. Results Among the 220 eligible patients, 133 were diagnosed with TB (men/women: 76/57; age range: 17–80 years, confirmed TB: 9, probable TB: 124). Forty-eight patients who were diagnosed with other specific diseases were assigned as negative controls, and 39 patients with indeterminate final diagnosis were excluded from statistical analysis. The sensitivity, specificity, PPV, NPV, and accuracy of histological AFB (HAFB) for the diagnosis of smear-negative were 61.7% (82/133), 100% (48/48), 100% (82/82), 48.5% (48/181), and 71.8% (130/181), respectively. The sensitivity, specificity, PPV, and NPV of histological PCR were 89.5% (119/133), 95.8% (46/48), 98.3% (119/121), and 76.7% (46/60), respectively, demonstrating that histological PCR had significantly higher accuracy (91.2% [165/181]) than histological acid-fast staining (71.8% [130/181]), P < 0.001. Parallel testing of histological AFB

  6. 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

    NASA Astrophysics Data System (ADS)

    Zhu, Xiao; Xing, Da

    2012-12-01

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

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

    PubMed

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

    2015-03-03

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

  8. Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

    PubMed

    Xu, Yao; Zheng, Zhi

    2016-05-15

    We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate.

  9. Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

    PubMed

    Machado, R L; Garret, D O; Adagu, I S; Warhurst, D C; Póvoa, M M

    1998-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

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

  11. Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP)

    PubMed Central

    Poole, Catherine B.; Li, Zhiru; Alhassan, Andy; Guelig, Dylan; Diesburg, Steven; Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C.; LaBarre, Paul; Wanji, Samuel; Burton, Robert A.; Carlow, Clotilde K. S.

    2017-01-01

    Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs. PMID:28199317

  12. A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

    PubMed Central

    LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

    2011-01-01

    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. PMID:21573065

  13. A label-free signal amplification assay for DNA detection based on exonuclease III and nucleic acid dye SYBR Green I.

    PubMed

    Zheng, Aihua; Luo, Ming; Xiang, Dongshan; Xiang, Xia; Ji, Xinghu; He, Zhike

    2013-09-30

    We have developed a new fluorescence method for specific single-stranded DNA sequences with exonuclease III (Exo III) and nucleic acid dye SYBR Green I. It is demonstrated by a reverse transcription oligonucleotide sequence (target DNA, 27 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of the target DNA, the hairpin-probe is in the stem-closed structure, the fluorescence of SYBR Green I is very strong. In the presence of the target DNA, the hairpin-probe hybridizes with the target DNA to form double-stranded structure with a blunt 3'-terminus. Thus, in the presence of Exo III, only the 3'-terminus of probe is subjected to digestion. Exo III catalyzes the stepwise removal of mononucleotides from this terminus, releasing the target DNA. The released target DNA then hybridizes with another probe, whence the cycle starts anew. The signal of SYBR Green I decreases greatly. This system provides a detection limit of 160 pM, which is comparable to the existing signal amplification methods that utilized Exo III as a signal amplification nuclease. Due to the unique property of Exo III, this method shows excellent detection selectivity for single-base discrimination. More importantly, superiors to other methods based on Exo III, these probes have the advantages of easier to design, synthesize, purify and thus are much cheaper and more applicable. This new approach could be widely applied to sensitive and selective nucleic acids detection.

  14. Biomaterials in light amplification

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  15. Enzymatic amplification-free nucleic acid hybridisation sensing on nanostructured thick-film electrodes by using covalently attached methylene blue.

    PubMed

    García-González, Raquel; Costa-García, Agustín; Fernández-Abedul, M Teresa

    2015-09-01

    Amplification-free (referring to enzymatic amplification step) detection methodologies are increasing in biosensor development due to the need of faster and simpler protocols. However, for maintaining sensitivity without this step, highly detectable molecules or very sensitive detection techniques are required. The nanostructuration of transducer surfaces with carbon nanotubes (CNTs), gold nanoparticles (AuNPs) or both in nanohybrid configurations has been employed in this work for DNA hybridisation sensing purposes. Methylene blue (MB), covalently attached to single stranded DNA, (ssDNA) was incubated with a complementary sequence immobilized on nanostructured screen-printed electrodes (AuSPEs). Although CNTs can increase notoriously the signal of the marker, adsorptive properties should also be considered when bioassays are performed because non-specific adsorption (NSA) phenomena are magnified. In this work, strategies for decreasing NSA were thoroughly evaluated for the detection of Mycoplasma pneumoniae (MP) on CNTs-nanostructured screen-printed electrodes. Among them, the employ of UV-radiation or long incubation times (72h) allowed obtaining higher signals for the complementary strand with respect to the non-complementary one. The use of CNTs/AuNPs nanohybrids, together with the use of streptavidin-biotin (ST-B) interaction allows the higher differentiation (with a 3.5 ratio) in the genosensing of M. pneumoniae.

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

    PubMed

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

    2015-08-01

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

  17. Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

    PubMed

    Kubota, Ryo; Labarre, Paul; Weigl, Bernhard H; Li, Yong; Haydock, Paul; Jenkins, Daniel M

    2013-04-01

    We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.

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

    PubMed

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

    2016-04-15

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

  19. Detection of trace amounts of target DNA from massive background of nucleic acids by using LM-PCR-based pre-amplification method.

    PubMed

    Pan, Xiaoming; Wang, Jing; Zhang, Yanfang; Dong, Ping; Li, Chunchuan; Liang, Xingguo

    2016-11-08

    The sensitivity and specificity of DNA detection may decrease when the target DNA is in very low abundance. To effectively detect trace amounts of target DNA from massive background of nucleic acids, we have developed a powerful multiplex pre-amplification method based on ligation-mediated PCR (LM-PCR) that can greatly enrich multiple target DNAs from massive backgrounds. By employing type IIS restriction endonuclease (REase) and specifically designed oligonucleotide adapters, target DNA can be pre-amplified with high efficiency and sensitivity. Combining with normal PCR, ten copies of target DNA was effectively detected from over 10(8) times more excessive backgrounds with high specificity and ten times more effectively than conventional PCR. In particular, the usage of universal primer in the pre-amplification PCR (pre-amp PCR) ensured that multiple targets could be equivalently amplified, which was confirmed by quantitative PCR (qPCR), indicating it could meet the demands of high-throughput detection. The flexibility and applicability of pre-amp PCR was validated by using different microorganisms DNA as targets and employing two different type IIS REases. The results suggest that the pre-amp PCR method has broad application prospects in various gene detection fields. This article is protected by copyright. All rights reserved.

  20. Nucleic acid amplification tests (polymerase chain reaction, ligase chain reaction) for the diagnosis of Chlamydia trachomatis and Neisseria gonorrhoeae in pediatric emergency medicine.

    PubMed

    Corneli, Howard M

    2005-04-01

    Nucleic acid amplification tests, such as ligase chain reaction and polymerase chain reaction, offer potential advantages of speed, simplicity, and accuracy in the detection of genitourinary tract infection with Neisseria gonorrhoeae and Chlamydia trachomatis. Their appropriate use in pediatric emergency medicine depends on an understanding of their strengths and weaknesses. Problems arise in defining the sensitivity and, especially, specificity of these tests. The clinical scenario, the site of infection, the age and sex of the patient, and especially the presence or absence of medicolegal concerns strongly affect the applicability of these tests. The risk of false positives may be significant even when legal concerns do not arise and even if a highly specific test is used. This article reviews the uses and limitations of such tests in pediatric emergency medicine. Discussion is directed to both technical and practical considerations.

  1. A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds.

    PubMed Central

    Pérez-Llarena, F J; Liras, P; Rodríguez-García, A; Martín, J F

    1997-01-01

    A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid. PMID:9068654

  2. Structure and sequence based analysis of alpha-amylase evolution.

    PubMed

    Singh, Swati; Guruprasad, Lalitha

    2014-01-01

    α-Amylases hydrolyze α- 1,4-glycosidic bonds during assimilation of biological macromolecules. The amino acid sequences of these enzymes in thousands of diverse organisms are known and the 3D structures of several proteins have been solved. The 3D structure analysis of these universal enzymes from diverse organisms has been studied by the generation of phylogenetic trees and structure based sequence analysis to generate a metric for the degree of conservation that is responsible for individual speciation. Greater similarities are observed between reference NCBI tree and structure based phylogenetic tree compared to sequence based phylogenetic tree indicating that structures truly represent the functional aspects of proteins than from the sequence information alone. We report differences in the profile specific conserved and insertion/deletion regions, factors responsible for the Ca(2+) and Cl(-) ion binding and the disulfide connectivity pattern that discriminate the enzymes over evolution.

  3. Monte Carlo Modeling-Based Digital Loop-Mediated Isothermal Amplification on a Spiral Chip for Absolute Quantification of Nucleic Acids.

    PubMed

    Xia, Yun; Yan, Shuangqian; Zhang, Xian; Ma, Peng; Du, Wei; Feng, Xiaojun; Liu, Bi-Feng

    2017-03-21

    Digital loop-mediated isothermal amplification (dLAMP) is an attractive approach for absolute quantification of nucleic acids with high sensitivity and selectivity. Theoretical and numerical analysis of dLAMP provides necessary guidance for the design and analysis of dLAMP devices. In this work, a mathematical model was proposed on the basis of the Monte Carlo method and the theories of Poisson statistics and chemometrics. To examine the established model, we fabricated a spiral chip with 1200 uniform and discrete reaction chambers (9.6 nL) for absolute quantification of pathogenic DNA samples by dLAMP. Under the optimized conditions, dLAMP analysis on the spiral chip realized quantification of nucleic acids spanning over 4 orders of magnitude in concentration with sensitivity as low as 8.7 × 10(-2) copies/μL in 40 min. The experimental results were consistent with the proposed mathematical model, which could provide useful guideline for future development of dLAMP devices.

  4. A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe₃O₄@Au nanoparticles with graphene sheet.

    PubMed

    Liu, Meiling; Chen, Qiong; Lai, Cailang; Zhang, Youyu; Deng, Jianhui; Li, Haitao; Yao, Shouzhuo

    2013-10-15

    A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid (AA), dopamine (DA), uric acid (UA) and acetaminophen (AC) was fabricated by a nanocomposite of ferrocene thiolate stabilized Fe₃O₄@Au nanoparticles with graphene sheet. The platform was constructed by coating a newly synthesized phenylethynyl ferrocene thiolate (Fc-SAc) modified Fe₃O₄@Au NPs coupling with graphene sheet/chitosan (GS-chitosan) on a glassy carbon electrode (GCE) surface. The Fe₃O₄@Au-S-Fc/GS-chitosan modified GCE exhibits a synergistic catalytic and amplification effect toward AA, DA, UA and AC oxidation. The oxidation peak currents of the four compounds on the electrode were linearly dependent on AA, DA, UA and AC concentrations in the ranges of 4-400 μM, 0.5-50 μM, 1-300 μM and 0.3-250 μM in the individual detection of each component, respectively. By simultaneously changing the concentrations of AA, DA, UA and AC, their electrochemical oxidation peaks appeared at -0.03, 0.15, 0.24 and 0.35 V, and good linear current responses were obtained in the concentration ranges of 6-350, 0.5-50, 1-90 and 0.4-32 μM with the detection limits of 1, 0.1, 0.2 and 0.05 μM (S/N=3), respectively.

  5. Enrichment, amplification, and sequence-based typing of Salmonella enterica and other foodborne pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection and characterization of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture; this is ideally followed by strain typing which provides information critical to outbreak and source investigations. Pulsed-field gel e...

  6. An enrichment, amplification, and sequence-based typing (EAST) approach for foodborne pathogen detection and surveillance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Detection of foodborne pathogens typically involves microbiological enrichment with subsequent isolation and identification of a pure culture. This is typically followed by strain typing, which provides information critical to outbreak and source investigations. In the early 1990’s pul...

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

    PubMed Central

    Gulliksen, Anja; Keegan, Helen; Martin, Cara; O'Leary, John; Solli, Lars A.; Falang, Inger Marie; Grønn, Petter; Karlgård, Aina; Mielnik, Michal M.; Johansen, Ib-Rune; Tofteberg, Terje R.; Baier, Tobias; Gransee, Rainer; Drese, Klaus; Hansen-Hagge, Thomas; Riegger, Lutz; Koltay, Peter; Zengerle, Roland; Karlsen, Frank; Ausen, Dag; Furuberg, Liv

    2012-01-01

    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. PMID:22235204

  8. [Viral safety of biologicals: evaluation of hepatitis C virus (HCV) nucleic acid amplification test (NAT) assay and development of concentration method of HCV for sensitive detection by NAT].

    PubMed

    Uchida, Eriko; Yamaguchi, Teruhide

    2010-02-01

    The most important issue for the safety of biological products and blood products derived from human sources is how to prevent transmission of infectious agents. The hepatitis C virus (HCV) is a major public health problem due to its high prevalence. HCV is mainly transmitted by exposure to blood and highly infectious during the early window period with extremely low viral loads. Therefore it is important to develop more sensitive detection methods for HCV. In the case of blood products, both serological test and nucleic acid amplification test (NAT) are required to detect HCV. Since NAT is highly sensitive, establishment of a new standard is required for validation of NAT assay. NAT guideline and establishment of the standard for HCV RNA and HCV genotype panel is introduced in this review. On the other hand, to enhance the sensitivity of virus detection by NAT, a novel viral concentration method using polyethyleneimine (PEI)-conjugated magnetic beads (PEI beads) was developed. PEI beads concentration method is applicable to a wide range of viruses including HCV. Studies using the national standard for HCV RNA, HCV genotype panel and seroconversion panel, suggest that virus concentration method using PEI-beads is useful for improvement of the sensitivity of HCV detection by NAT and applicable to donor screening for HCV.

  9. Screening of organ and tissue donors for West Nile virus by nucleic acid amplification--a three year experience in Alberta.

    PubMed

    Tilley, Peter A G; Fox, Julie D; Lee, Bonita; Chui, Linda; Preiksaitis, Jutta

    2008-10-01

    West Nile Virus (WNV)-specific nucleic acid amplification testing (NAAT) of organ and tissue donors remains controversial. We report three years of WNV donor screening in Alberta Canada using NAAT. Between 2003 and 2005, 1549 initial specimens were received. A valid negative result was issued within the specified turnaround time on 1531 (98.8%). The initial NAAT was successful for 1393 samples (90%), while repeat testing using an alternate NAAT resolved a further 126 samples. For 12 of 14 donors, a second specimen provided a valid negative result. Failure to generate a valid negative result in time resulted in rescheduling of one living related organ transplant, and surgery proceeded in the absence of a final result in one multi-organ donation after risk assessment. For 11 tissue donors, tissues were discarded due to lack of a WNV result. Invalid results usually occurred on postmortem haemolyzed tissue donor samples due to inhibitory reactions. There were no confirmed positive donors, no false-positive results and no solid organs lost due to WNV testing. We conclude that WNV NAAT of organ and tissue donors can be implemented without compromising availability of donors but requires committed laboratory support.

  10. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    PubMed Central

    2010-01-01

    Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum PMID:21143837

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

    PubMed

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

    2016-10-01

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

  12. Detecting asymptomatic Trichomonas vaginalis in females using the BD ProbeTec™ Trichomonas vaginalis Q(x) nucleic acid amplification test.

    PubMed

    Lord, Emily; Newnham, Tana; Dorrell, Lucy; Jesuthasan, Gerald; Clarke, Lorraine; Jeffery, Katie; Sherrard, Jackie

    2017-03-01

    Trichomonas vaginalis (TV) rates in women are increasing and many are asymptomatic. Nucleic acid amplification tests (NAATs) are becoming the 'gold standard' for diagnosis. We aimed to establish our asymptomatic TV rates by testing all women attending Oxfordshire's Sexual Health service, regardless of symptoms, using the BD ProbeTec™ TV Q(x) NAATs (BDQ(x)). During BDQ(x)'s verification process, the sensitivity and specificity were calculated using results of 220 endocervical samples from symptomatic women, compared with culture. BDQ(x) was subsequently implemented and prospectively evaluated over 6 months in female attendees. Wet mount microscopy was also performed in symptomatics. Demographic and clinical characteristics of those diagnosed were analysed. From 220 samples tested by BDQ(x) and culture: 5 were positive on both and one solely using BDQ(x), giving a sensitivity and specificity of 100% and 99.53%, respectively. In the prospective cohort, of 5775 BDQ(x) tests, 33 (0.57%) were positive. 11/33 (33%) patients were asymptomatic. All patients diagnosed had risk factors: age >25 years (85%), residence in a deprived area (79%) and black ethnicity (21%). Despite BDQ(x) being highly sensitive and specific, with our low TV prevalence universal screening may not be justified. Targeted screening using local demographic data merits further investigation.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-01-21

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

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

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  16. Chemical amplification based on fluid partitioning

    DOEpatents

    Anderson, Brian L.; Colston, Jr., Billy W.; Elkin, Chris

    2006-05-09

    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.

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

    PubMed

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

    2012-05-01

    Dry-shipped and mailed vaginal swabs collected at home have been used in research studies for the detection of Chlamydia trachomatis (CT), Neisseria 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 US mail. 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 100 μL of dilutions and 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. All paired dry and wet swabs were detectable for CT. For GC, all paired dry and wet swabs were detectable for GC at concentrations ≥ 10(3). At 10(2) and 10 CFU/mL, the 10 replicate GC results were variably positive. For TV, wet and dry shipped concentrations >10(2) 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. 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.

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

    PubMed

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

    2016-02-01

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

  19. Comparison of the Luminex xTAG respiratory viral panel with in-house nucleic acid amplification tests for diagnosis of respiratory virus infections.

    PubMed

    Pabbaraju, Kanti; Tokaryk, Kara L; Wong, Sallene; Fox, Julie D

    2008-09-01

    Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel (RVP) assay with individual real-time NATs used at the Provincial Laboratory of Public Health, Calgary, Alberta, Canada. The study included 1,530 specimens submitted for diagnosis of respiratory infections from December 2006 to May 2007. Direct-fluorescent-antigen-positive nasopharyngeal samples were excluded from this study. A total of 690 and 643 positives were detected by RVP and in-house NATs, respectively. Kappa correlation between in-house NATs and RVP for all targets ranged from 0.721 to 1.000. The majority of specimens missed by in-house NATs (96.7%) were positive for picornaviruses. Samples missed by RVP were mainly positive for adenovirus (51.7%) or respiratory syncytial virus (27.5%) by in-house NATs and in general had low viral loads. RVP allows for multiplex detection of 20 (and differentiation between 19) respiratory virus targets with considerable time and cost savings compared with alternative NATs. Although this first version of the RVP assay has lower sensitivity than in-house NATs for detection of adenovirus, it has good sensitivity for other targets. The identification of picornaviruses and coronaviruses and concurrent typing of influenza A virus by RVP, which are not currently included in our diagnostic testing algorithm, will improve our diagnosis of respiratory tract infections.

  20. Establishment of the 1st World Health Organization International Standard for Plasmodium falciparum DNA for nucleic acid amplification technique (NAT)-based assays

    PubMed Central

    Padley, David J; Heath, Alan B; Sutherland, Colin; Chiodini, Peter L; Baylis, Sally A

    2008-01-01

    Background In order to harmonize results for the detection and quantification of Plasmodium falciparum DNA by nucleic acid amplification technique (NAT)-based assays, a World Health Organization (WHO) collaborative study was performed, evaluating a series of candidate standard preparations. Methods Fourteen laboratories from 10 different countries participated in the collaborative study. Four candidate preparations based upon blood samples parasitaemic for P. falciparum were evaluated in the study. Sample AA was lyophilized, whilst samples BB, CC and DD were liquid/frozen preparations. The candidate standards were tested by each laboratory at a range of dilutions in four independent assays, using both qualitative and quantitative NAT-based assays. The results were collated and analysed statistically. Results Twenty sets of data were returned from the participating laboratories and used to determine the mean P. falciparum DNA content for each sample. The mean log10 "equivalents"/ml were 8.51 for sample AA, 8.45 for sample BB, 8.35 for sample CC, and 5.51 for sample DD. The freeze-dried preparation AA, was examined by accelerated thermal degradation studies and found to be highly stable. Conclusion On the basis of the collaborative study, the freeze-dried material, AA (NIBSC code No. 04/176) was established as the 1st WHO International Standard for P. falciparum DNA NAT-based assays and has been assigned a potency of 109 International Units (IU) per ml. Each vial contains 5 × 108 IU, equivalent to 0.5 ml of material after reconstitution. PMID:18652656

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Liu, Yi; Duan, Chunhong; Zhang, Chunxiu; Yang, Xiaomeng; Zhao, Yan; Dong, Rui; Zhou, Jiajing; Gai, Zhongtao

    2015-01-01

    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.

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

    PubMed

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

    2014-09-22

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

  4. 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.

    PubMed

    Verkooyen, Roel P; Noordhoek, Gerda T; Klapper, Paul E; Reid, Jim; Schirm, Jurjen; Cleator, Graham M; Ieven, Margareta; Hoddevik, Gunnar

    2003-07-01

    The 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, including three negative, two strongly positive, and five weakly positive samples. Ninety-six laboratories in 22 countries participated with a total of 102 data sets. Of 204 strongly positive samples 199 (97.5%) were correctly reported, and of 506 weakly positive samples 466 (92.1%) were correctly reported. In 74 (72.5%) data sets correct results were reported on all samples, and 17 data sets (16.7%) showed either one false-negative or one false-positive result. In another 11 data sets, two or more incorrect results were reported, and two data sets reported a false-positive result on one negative sample. The Roche COBAS Amplicor test was performed in 44 (43%) data sets, the Abbott LCx assay was performed in 31 (30%) data sets, the Roche Amplicor manual assay was performed in 9 (9%) data sets, an in-house PCR was performed in 9 (9%) data sets, the Becton Dickinson ProbeTec ET assay was performed in 5 (4.9%) data sets, and the GenProbe TMA assay was performed in 4 (3.9%) data sets. The results of the Roche Amplicor manual (95.6% correct), COBAS Amplicor (97.0%), and Abbott LCx (94.8%) tests were comparable (P = 0.48). The results with the in-house PCR, BD ProbeTec ET, and GenProbe TMA tests were reported correctly in 88.6, 98, and 92.5% of the tests, respectively. Freeze-drying of clinical urine specimens proved to be a successful method for generating standardized, stable, and easy-to-transport samples for the detection of C. trachomatis by using NATs. Although the results, especially the specificity, for this proficiency panel were better than most quality control studies, sensitivity problems occurred frequently, underlining the need for good laboratory practice and reference reagents to monitor the

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

    PubMed

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

    2017-03-08

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

  6. Isothermal amplification coupled with rapid flow-through hybridisation for sensitive diagnosis of Plum pox virus.

    PubMed

    Olmos, Antonio; Bertolini, Edson; Cambra, Mariano

    2007-01-01

    A nucleic acid sequence-based amplification method coupled with rapid flow-through hybridisation (NASBA-FH) was developed for diagnosis of Plum pox virus (PPV). The sensitivity level achieved by NASBA-FH was 10 times higher than that obtained by Co-PCR and 1000 times higher than the sensitivity afforded by RT-PCR. In addition, samples from 262 stone-fruit trees collected during winter and spring seasons were analysed. These samples were tested using methods recommended by the European and Mediterranean Plant Protection Organization to detect PPV (DASI-ELISA, RT-PCR and Co-PCR) and by NASBA-FH. Winter PPV diagnostic results by ELISA and NASBA-FH coincided in 90.8%, while ELISA and PCR-based methods coincided in 91.6% and PCR-based methods with NASBA-FH agreed in 95.4%. In spring, diagnostic results were similar with all the molecular techniques, which agreed with ELISA results for 98.8% of the trees. NASBA-FH was able to detect more positive infections in winter, which were later confirmed in spring. These results indicate that NASBA-FH is a suitable molecular method for routine PPV detection in the winter and spring. This user-friendly isothermal RNA amplification coupled with a very fast flow-through hybridisation (15 min) opens up new possibilities for rapid and reliable diagnosis of a variety of pathogens.

  7. Early amplification options.

    PubMed

    Gabbard, Sandra Abbott; Schryer, Jennifer

    2003-01-01

    Children with permanent hearing loss have been remediated with hearing amplification devices for decades. The influx of young infants identified with hearing loss through successful newborn hearing screening programs has established a need for amplification resources for infants within the first six months of life. For the approximately two of every 1000 infants born who are identified with bilateral hearing loss [Mehl and Thomson, 1998, Pediatrics 101, p. e4], the use of amplification is commonly the first step in treating the sequella of their loss. The use of hearing aids, combined with early intervention, has been shown to significantly improve the speech and language skills of young children with hearing loss [Yoshinaga-Itano, 2000, Seminars in Hearing 21, p. 309]. Speech and language delays have contributed to compromised academic performance of school aged children with hearing loss [Johnson et al., 1997, Educational Audiology Handbook, Singular Publishing, San Diego]. Most hard-of-hearing and deaf children use hearing aids and other assistive listening devices every day throughout their lifetime and the life expectancy of a hearing aid is only five to eight years. The current challenge for pediatric audiologists is selecting and evaluating the available amplification to provide the best options for children and their families. Amplification technology has seen an explosion in growth the past few years and the options continue to expand rapidly. This article examines currently available amplification technology and reviews the selection criteria that may be used for infants and young children. Issues such as style, type, amplification features, signal processing strategies, and verification and validation tools are also discussed.

  8. Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

    PubMed

    Lamas-Maceiras, Mónica; Vaca, Inmaculada; Rodríguez, Esther; Casqueiro, Javier; Martín, Juan F

    2006-04-01

    A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  10. Chemical amplification based on fluid partitioning in an immiscible liquid

    DOEpatents

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

    2010-09-28

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

  11. High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum.

    PubMed

    Xie, Hehuang; Wang, Min; Bonaldo, Maria de F; Smith, Christina; Rajaram, Veena; Goldman, Stewart; Tomita, Tadanori; Soares, Marcelo B

    2009-07-01

    DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5' flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer.

  12. Sequence-Based Classification Scheme for the Genus Legionella Targeting the mip Gene

    PubMed Central

    Ratcliff, Rodney M.; Lanser, Janice A.; Manning, Paul A.; Heuzenroeder, Michael W.

    1998-01-01

    The identification and speciation of strains of Legionella is often difficult, and even the more successful chromatographic classification techniques have struggled to discriminate newly described species. A sequence-based genotypic classification scheme is reported, targeting approximately 700 nucleotide bases of the mip gene and utilizing gene amplification and direct amplicon sequencing. With the exception of Legionella geestiana, for which an amplicon was not produced, the scheme clearly and unambiguously discriminated among the remaining 39 Legionella species and correctly grouped 26 additional serogroup and reference strains within those species. Additionally, the genotypic classification of approximately 150 wild strains from several continents was consistent with their phenotypic classification, with the exception of a few strains where serological cross-reactivity was complex, potentially confusing the latter classification. Strains thought to represent currently uncharacterized species were also found to be genotypically unique. The scheme is technically simple for a laboratory with even basic molecular capabilities and equipment, if access to a sequencing laboratory is available. PMID:9620377

  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.

    PubMed

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

    2016-07-15

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

  14. Questioning cochlear amplification

    NASA Astrophysics Data System (ADS)

    van der Heijden, Marcel; Versteegh, Corstiaen P. C.

    2015-12-01

    Thirty years ago it was hypothesized that motile processes inject mechanical energy into cochlear traveling waves. This mechanical amplification, alternatively described as negative damping, is invoked to explain both the sensitivity and the nonlinear compression of cochlear responses. There is a recent trend to present cochlear amplification as an established fact, even though the evidence is at most circumstantial and several thorny problems have remained unresolved. We analyze several of these issues, and present new basilar membrane recordings that allowed us to quantify cochlear energy flow. Specifically, we address the following questions: (1) Does auditory sensitivity require narrowband amplification? (2) Has the "RC problem" (lowpass filtering of outer hair cell receptor potential) been resolved? (3) Can OHC motility improve auditory sensitivity? (4) Is there a net power gain between neighboring locations on the basilar membrane? The analyses indicate that mechanical amplification in the cochlea is neither necessary nor useful, and that realizing it by known forms of motility would reduce sensitivity rather than enhance it. Finally, our experimental data show that the peaking of the traveling wave is realized by focusing the acoustic energy rather than amplifying it. (Abbreviations. BM: basilar membrane; CF: characteristic frequency; IHC: inner hair cell; ME: middle ear; MT; mechanotransducer; OHC: outer hair cell; SPL: sound pressure level.)

  15. Sequence-based classification and identification of Fungi.

    PubMed

    Hibbett, David; Abarenkov, Kessy; Koljalg, Urmas; Opik, Maarja; Chai, Benli; Cole, James R; Wang, Qiong; Crous, Pedro W; Robert, Vincent A R G; Helgason, Thorunn; Herr, Josh; Kirk, Paul; Lueschow, Shiloh; O'Donnell, Kerry; Nilsson, Henrik; Oono, Ryoko; Schoch, Conrad L; Smyth, Christopher; Walker, Donny; Porras-Alfaro, Andrea; Taylor, John W; Geiser, David M

    2016-10-19

    Fungal taxonomy and ecology have been revolutionized by the application of molecular methods and both have increasing connections to genomics and functional biology. However, data streams from traditional specimen- and culture-based systematics are not yet fully integrated with those from metagenomic and metatranscriptomic studies, which limits understanding of the taxonomic diversity and metabolic properties of fungal communities. This article reviews current resources, needs, and opportunities for sequence-based classification and identification (SBCI) in fungi as well as related efforts in prokaryotes. To realize the full potential of fungal SBCI it will be necessary to make advances in multiple areas. Improvements in sequencing methods, including long-read and single-cell technologies, will empower fungal molecular ecologists to look beyond ITS and current shotgun metagenomics approaches. Data quality and accessibility will be enhanced by attention to data and metadata standards and rigorous enforcement of policies for deposition of data and workflows. Taxonomic communities will need to develop best practices for molecular characterization in their focal clades, while also contributing to globally useful datasets including ITS. Changes to nomenclatural rules are needed to enable valid publication of sequence-based taxon descriptions. Finally, cultural shifts are necessary to promote adoption of SBCI and to accord professional credit to individuals who contribute to community resources.

  16. Qualitative detection of avian influenza A (H5N1) viruses: a comparative evaluation of four real-time nucleic acid amplification methods.

    PubMed

    Chantratita, Wasun; Sukasem, Chonlaphat; Kaewpongsri, Supaporn; Srichunrusami, Chutatip; Pairoj, Wantanit; Thitithanyanont, Arunee; Chaichoune, Kridsada; Ratanakron, Parntep; Songserm, Thaweesak; Damrongwatanapokin, Sudarat; Landt, Olfert

    2008-01-01

    The aim of this study was to determine the performance of real-time amplification based methods - NASBA, TaqMan, RT-FRET, and RT-PCR LUXtrade mark formats - for the detection of influenza A (H5N1) virus RNA. In an analysis of 54 samples obtained from a range of animal species in Thailand during the period 2003-2006, results showed that the NASBA (H5=98.2%, N1=96.3%), TaqMan (H5=98.2%, N1=96.3%) and FRET (H5=98.2%, N1=96.3%) had significantly higher rates of positive detection than LUX (H5=94.4%, N1=50.0%; P<0.001) for influenza A, H5 and N1 isolates. There were no false-positive results from any methods used in the negative-control group of samples. The limits of analytical detection were at least 10copies/reaction in real-time NASBA and LUX assays, while FRET and TaqMan assay appeared to be less sensitive at > or =100copies/reaction. The assays were relatively specific without cross-reactivity to a number of other influenza strains or viral pathogens. In conclusion, our study demonstrated that real-time NASBA, TaqMan and FRET assays can be used to detect influenza A (H5N1) from a wide range of hosts, and be specific for H5N1 samples obtained during different outbreaks (2003-2006). All assays provided the benefit of rapid influenza H5N1 identification for early diagnosis, in the range of hours, and they are well suited to high throughput analyses.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27600711

  18. Head-to-head comparison of second-generation nucleic acid amplification tests for detection of Chlamydia trachomatis and Neisseria gonorrhoeae on urine samples from female subjects and self-collected vaginal swabs.

    PubMed

    Chernesky, Max; Jang, Dan; Gilchrist, Jodi; Hatchette, Todd; Poirier, André; Flandin, Jean-Frederic; Smieja, Marek; Ratnam, Sam

    2014-07-01

    In a comparison of 4 second-generation nucleic acid amplification tests performed with self-collected vaginal swab (SCVS) and first-void urine (FVU) specimens from 575 women, SCVS specimens indicated more infections than did FVU specimens in all assays. The prevalence rates were 9% (53/575 patients) for Chlamydia trachomatis and 2% (11/575 patients) for Neisseria gonorrhoeae. The clinical sensitivities for testing SCVS specimens for C. trachomatis were 98.1% on a Tigris system and 96.2% on a Panther system for the Aptima Combo 2 assay (Hologic Gen-Probe), 98.0% for the RealTime CT/NG assay on an m2000 instrument (Abbott), 90.6% for the ProbeTec CT/GC Q(x) assay on the Viper system (Becton Dickinson), and 84.6% for the cobas CT/NG assay on the cobas 4800 platform (Roche). Clinical sensitivities for C. trachomatis in FVU specimens were 88.7% (Tigris) and 88.0% (Panther) for the Aptima Combo 2 assay, 76.9% for the RealTime CT/NG assay, 75.5% for the ProbeTec CT/GC Q(x) assay, and 81.1% for the cobas CT/NG assay. Clinical sensitivities of the assays for N. gonorrhoeae, with limited positive results, ranged from 63.6% to 100%. Specificities for both infections ranged from 98.4 to 100%. Differences in analytical sensitivities and levels of molecular targets in clinical samples but not inhibitors of amplification may explain the differences in clinical sensitivities.

  19. 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.

    PubMed

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

    2016-09-29

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

  20. Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

    PubMed

    Metcalf, Gavin A D; Shibakawa, Akifumi; Patel, Hinesh; Sita-Lumsden, Ailsa; Zivi, Andrea; Rama, Nona; Bevan, Charlotte L; Ladame, Sylvain

    2016-08-16

    Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening.

  1. Species specific identification of spore-producing microbes using the gene sequence of small acid-soluble spore coat proteins for amplification based diagnostics

    DOEpatents

    McKinney, Nancy

    2002-01-01

    PCR (polymerase chain reaction) primers for the detection of certain Bacillus species, such as Bacillus anthracis. The primers specifically amplify only DNA found in the target species and can distinguish closely related species. Species-specific PCR primers for Bacillus anthracis, Bacillus globigii and Clostridium perfringens are disclosed. The primers are directed to unique sequences within sasp (small acid soluble protein) genes.

  2. Gravitomagnetic amplification in cosmology

    SciTech Connect

    Tsagas, Christos G.

    2010-02-15

    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 gravitomagnetic 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 gravitomagnetic interaction and discuss its potential implications.

  3. Isothermal Multiple Displacement Amplification

    PubMed Central

    Luthra, Rajyalakshmi; Medeiros, L. Jeffrey

    2004-01-01

    Isothermal multiple strand displacement amplification (IMDA) of the whole human genome is a promising method for procuring abundant DNA from valuable and often limited clinical specimens. However, whether DNA generated by this method is of high quality and a faithful replication of the DNA in the original specimen, allowing for subsequent molecular diagnostic testing, requires verification. In this study, we evaluated the suitability of IMDA-generated DNA (IMDA-DNA) for detecting antigen receptor gene rearrangements, chromosomal translocations, and gene mutations using Southern blot analysis, polymerase chain reaction (PCR) methods, or sequencing methods in 28 lymphoma and leukemia clinical specimens. Molecular testing before and after whole genome amplification of these specimens using the IMDA technique showed concordance in 27 of 28 (96%) specimens. Analysis of IMDA-DNA by Southern blot analysis detected restriction fragments >12 kilobases long. No amplification bias was observed at all loci tested demonstrating that this method can be useful in generating large amounts of unbiased, high molecular weight DNA from limited clinical specimens. PMID:15269301

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

    PubMed

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

    2016-05-01

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

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

    PubMed Central

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

    2016-01-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease. PMID:27284221

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

    PubMed

    Yang, Jing; Xu, Xinxin; Liu, Gang

    2012-11-20

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

  7. Mass spectrometry signal amplification for ultrasensitive glycoprotein detection using gold nanoparticle as mass tag combined with boronic acid based isolation strategy.

    PubMed

    Liu, Minbo; Zhang, Lijuan; Xu, Yawei; Yang, Pengyuan; Lu, Haojie

    2013-07-25

    We describe a novel method for rapid and ultrasensitive detection of intact glycoproteins without enzymatic pretreatment which was commonly used in proteomic research. This method is based on using gold nanoparticle (AuNP) as signal tag in laser desorption/ionization mass spectrometry (LDI-MS) analysis combined with boronic acid assisted isolation strategy. Briefly speaking, target glycoproteins were firstly isolated from sample solution with boronic acid functionalized magnetic microparticles, and then the surface modified gold nanoparticles were added to covalently bind to the glycoproteins. After that, these AuNP tagged glycoproteins were eluted from magnetic microparticles and applied to LDI-MS analysis. The mass signal of AuNP rather than that of glycoprotein was detected and recorded in this strategy. Through data processing of different standard glycoproteins, we have demonstrated that the signal of AuNP could be used to quantitatively represent glycoprotein. This method allows femtomolar detection of intact glycoproteins. We believe that the successful validation of this method on three different kinds of glycoproteins suggests the potential use for tracking trace amount of target glycoproteins in real biological samples in the near future.

  8. 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.

    PubMed

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

    2016-10-01

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

  9. Coherent white light amplification

    DOEpatents

    Jovanovic, Igor; Barty, Christopher P.

    2004-05-25

    A system for coherent simultaneous amplification of a broad spectral range of light that includes an optical parametric amplifier and a source of a seed pulse is described. A first angular dispersive element is operatively connected to the source of a seed pulse. A first imaging telescope is operatively connected to the first angular dispersive element and operatively connected to the optical parametric amplifier. A source of a pump pulse is operatively connected to the optical parametric amplifier. A second imaging telescope is operatively connected to the optical parametric amplifier and a second angular dispersive element is operatively connected to the second imaging telescope.

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

    PubMed

    Giuffrida, Maria Chiara; Spoto, Giuseppe

    2017-04-15

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

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

    PubMed

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

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

  12. Evidence of high-elevation amplification versus Arctic amplification

    PubMed Central

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961–2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction. PMID:26753547

  13. Arbitrary single primer amplification of trace DNA substrates yields sequence content profiles that are discriminatory and reproducible.

    PubMed

    Waters, James M; Eariss, Graham; Yeadon, P Jane; Kirkbride, K Paul; Burgoyne, Leigh A; Catcheside, David E A

    2012-02-01

    Single primer amplification is shown to yield a DNA profile that is reproducible when based on the sequence content of the amplicons rather than on the pattern of length polymorphism. The sequence-based profile increases in reliability with increasing numbers of cycles of amplification. This process uses an arbitrarily chosen primer and a low initial annealing temperature in order to amplify sequences from the whole metagenome present in a sample that may contain only trace DNA, and a large number of cycles to select subsets of sequences based on variable amplification efficiency. Using arrays, we demonstrate the utility and limitations of this approach for profiling the large metagenomes typical of soils and the trace DNA present in drug seizures. We suggest that this type of profiling will be most effective once next-generation sequencing and advanced sequence analysis becomes routine.

  14. Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylat...

  15. SFM: A novel sequence-based fusion method for disease genes identification and prioritization.

    PubMed

    Yousef, Abdulaziz; Moghadam Charkari, Nasrollah

    2015-10-21

    The identification of disease genes from human genome is of great importance to improve diagnosis and treatment of disease. Several machine learning methods have been introduced to identify disease genes. However, these methods mostly differ in the prior knowledge used to construct the feature vector for each instance (gene), the ways of selecting negative data (non-disease genes) where there is no investigational approach to find them and the classification methods used to make the final decision. In this work, a novel Sequence-based fusion method (SFM) is proposed to identify disease genes. In this regard, unlike existing methods, instead of using a noisy and incomplete prior-knowledge, the amino acid sequence of the proteins which is universal data has been carried out to present the genes (proteins) into four different feature vectors. To select more likely negative data from candidate genes, the intersection set of four negative sets which are generated using distance approach is considered. Then, Decision Tree (C4.5) has been applied as a fusion method to combine the results of four independent state-of the-art predictors based on support vector machine (SVM) algorithm, and to make the final decision. The experimental results of the proposed method have been evaluated by some standard measures. The results indicate the precision, recall and F-measure of 82.6%, 85.6% and 84, respectively. These results confirm the efficiency and validity of the proposed method.

  16. Sequence-Based Prediction of Type III Secreted Proteins

    PubMed Central

    Arnold, Roland; Brandmaier, Stefan; Kleine, Frederick; Tischler, Patrick; Heinz, Eva; Behrens, Sebastian; Niinikoski, Antti; Mewes, Hans-Werner; Horn, Matthias; Rattei, Thomas

    2009-01-01

    The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of ∼71% and selectivity of ∼85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will

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

    PubMed

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

    2016-07-01

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

  18. Self-primed isothermal amplification for genomic DNA detection of human papillomavirus.

    PubMed

    Lu, Wei; Yuan, Qingpan; Yang, Zhiliu; Yao, Bo

    2017-04-15

    Rolling circle amplification (RCA) is an isothermal amplification technique with high efficiency and perfect accuracy for nucleic acids detection. However, RCA technique suffers the limitation to detect short DNA or RNA molecules. For long nucleic acid molecules, enzymatic restriction as well as heat denaturation process is usually required, which makes the amplification not effective and strictly isothermal. In this article, a simple and efficient one-pot self-primed isothermal amplification (SIA) was developed for detection of genomic DNA directly based on the combination of nicking endonuclease assisted strand displacement amplification (SDA) and exponential RCA. In virtue of numerous nicking sites on the genome, a pre-amplification of the whole genome was performed through SDA with the specific cleaving of nicking endonuclease. Meanwhile, the single strand DNA with HPV target sequence generated from SDA could hybrid with the circle probe as a primer and trigger the exponential RCA as a result of the existence of nicking endonuclease. As the reaction temperature and enzyme were the same, the amplification could be operated in one pot. The reaction solution after amplification was added on the electrode for hybridization with the sulfydryl probe to achieve the electrochemical signal. Based on the isothermal amplification, genotyping of HPV 11, 16, 18 and the detection of HPV 18 in Hela cell line were attempted with satisfied results. This approach should be a promising tool for pathogene detection in clinical diagnostics and research.

  19. Experiments on the abiotic amplification of optical activity

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Blair, N. E.; Dirbas, F. M.

    1981-01-01

    Experiments concerning the physical mechanisms for the abiotic generation and chemical mechanisms for the amplification of optical activity in biological compounds are reviewed. Attention is given to experiments involving the determination of the differential adsorption of racemic amino acids on d- and l-quartz, the asymmetric photolysis of racemic amino acids by circularly polarized light, and the asymmetric radiolysis of solid amino acids by longitudinally polarized electrons, and the enantiomeric enrichments thus obtained are noted. Further experiments on the amplification of the chirality in the polymerization of D, L-amino acid mixtures and the hydrolysis of D-, L-, and D, L-polypeptides are discussed. It is suggested that a repetitive cycle of partial polymerization-hydrolyses may account for the abiotic genesis of optically enriched polypeptides on the primitive earth.

  20. [Recombinase Polymerase Amplification and its Applications in Parasite Detection].

    PubMed

    ZHENG, Wen-bin; WU, Yao-dong; MA, Jian-gang; ZHU, Xing-quan; ZHOU, Dong-hui

    2015-10-01

    Recombinase polymerase amplification (RPA) is a recently -developed isothermal nucleic-acid-amplification technology that is based on the nucleic acid replication mechanism in T4 bacteriophage. With this technique, nucleic-acid templates can be amplified to measurable levels within 20 min at 37-42 °C. The. RPA process has high sensitivity and specificity, and is simple to operate, thus nucleic acids can be detected rapidly in non-laboratory conditions. Since its development in 2006, the RPA technique has been applied in agriculture, food safety, medicine, transgene detection, etc. In this review, we will give an overview on the research progress of RPA and its application in parasite detection.

  1. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2001-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal. Specifically, the analyte transducer immobilized in a polymeric matrix can be a boronic acid moiety.

  2. Chromosomal destabilization during gene amplification.

    PubMed Central

    Ruiz, J C; Wahl, G M

    1990-01-01

    Acentric extrachromosomal elements, such as submicroscopic autonomously replicating circular molecules (episomes) and double minute chromosomes, are common early, and in some cases initial, intermediates of gene amplification in many drug-resistant and tumor cell lines. In order to gain a more complete understanding of the amplification process, we investigated the molecular mechanisms by which such extrachromosomal elements are generated and we traced the fate of these amplification intermediates over time. The model system consists of a Chinese hamster cell line (L46) created by gene transfer in which the initial amplification product was shown previously to be an unstable extrachromosomal element containing an inverted duplication spanning more than 160 kilobases (J. C. Ruiz and G. M. Wahl, Mol. Cell. Biol. 8:4302-4313, 1988). In this study, we show that these molecules were formed by a process involving chromosomal deletion. Fluorescence in situ hybridization was performed at multiple time points on cells with amplified sequences. These studies reveal that the extrachromosomal molecules rapidly integrate into chromosomes, often near or at telomeres, and once integrated, the amplified sequences are themselves unstable. These data provide a molecular and cytogenetic chronology for gene amplification in this model system; an early event involves deletion to generate extrachromosomal elements, and subsequent integration of these elements precipitates a cascade of chromosome instability. Images PMID:2188107

  3. Rational experiment design for sequencing-based RNA structure mapping.

    PubMed

    Aviran, Sharon; Pachter, Lior

    2014-12-01

    Structure mapping is a classic experimental approach for determining nucleic acid structure that has gained renewed interest in recent years following advances in chemistry, genomics, and informatics. The approach encompasses numerous techniques that use different means to introduce nucleotide-level modifications in a structure-dependent manner. Modifications are assayed via cDNA fragment analysis, using electrophoresis or next-generation sequencing (NGS). The recent advent of NGS has dramatically increased the throughput, multiplexing capacity, and scope of RNA structure mapping assays, thereby opening new possibilities for genome-scale, de novo, and in vivo studies. From an informatics standpoint, NGS is more informative than prior technologies by virtue of delivering direct molecular measurements in the form of digital sequence counts. Motivated by these new capabilities, we introduce a novel model-based in silico approach for quantitative design of large-scale multiplexed NGS structure mapping assays, which takes advantage of the direct and digital nature of NGS readouts. We use it to characterize the relationship between controllable experimental parameters and the precision of mapping measurements. Our results highlight the complexity of these dependencies and shed light on relevant tradeoffs and pitfalls, which can be difficult to discern by intuition alone. We demonstrate our approach by quantitatively assessing the robustness of SHAPE-Seq measurements, obtained by multiplexing SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemistry in conjunction with NGS. We then utilize it to elucidate design considerations in advanced genome-wide approaches for probing the transcriptome, which recently obtained in vivo information using dimethyl sulfate (DMS) chemistry.

  4. Chemical amplification of magnetic field effects relevant to avian magnetoreception

    NASA Astrophysics Data System (ADS)

    Kattnig, Daniel R.; Evans, Emrys W.; Déjean, Victoire; Dodson, Charlotte A.; Wallace, Mark I.; MacKenzie, Stuart R.; Timmel, Christiane R.; Hore, P. J.

    2016-04-01

    Magnetic fields as weak as the Earth's can change the yields of radical pair reactions even though the energies involved are orders of magnitude smaller than the thermal energy, kBT, at room temperature. Proposed as the source of the light-dependent magnetic compass in migratory birds, the radical pair mechanism is thought to operate in cryptochrome flavoproteins in the retina. Here we demonstrate that the primary magnetic field effect on flavin photoreactions can be amplified chemically by slow radical termination reactions under conditions of continuous photoexcitation. The nature and origin of the amplification are revealed by studies of the intermolecular flavin-tryptophan and flavin-ascorbic acid photocycles and the closely related intramolecular flavin-tryptophan radical pair in cryptochrome. Amplification factors of up to 5.6 were observed for magnetic fields weaker than 1 mT. Substantial chemical amplification could have a significant impact on the viability of a cryptochrome-based magnetic compass sensor.

  5. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, Stefan K.

    1998-01-01

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei.

  6. Sequence independent amplification of DNA

    DOEpatents

    Bohlander, S.K.

    1998-03-24

    The present invention is a rapid sequence-independent amplification procedure (SIA). Even minute amounts of DNA from various sources can be amplified independent of any sequence requirements of the DNA or any a priori knowledge of any sequence characteristics of the DNA to be amplified. This method allows, for example, the sequence independent amplification of microdissected chromosomal material and the reliable construction of high quality fluorescent in situ hybridization (FISH) probes from YACs or from other sources. These probes can be used to localize YACs on metaphase chromosomes but also--with high efficiency--in interphase nuclei. 25 figs.

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

    DOEpatents

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

    2012-05-08

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

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

    DOEpatents

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

    2015-06-02

    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.

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

    DOEpatents

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

    2017-02-28

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

  10. 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.

    PubMed

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

    2015-01-26

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

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

    PubMed

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

    2013-05-01

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

  12. Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

    PubMed Central

    2009-01-01

    A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA. PMID:21637462

  13. Microwave amplification with nanomechanical resonators.

    PubMed

    Massel, F; Heikkilä, T T; Pirkkalainen, J-M; Cho, S U; Saloniemi, H; Hakonen, P J; Sillanpää, M A

    2011-12-14

    The sensitive measurement of electrical signals is at the heart of modern technology. According to the principles of quantum mechanics, any detector or amplifier necessarily adds a certain amount of noise to the signal, equal to at least the noise added by quantum fluctuations. This quantum limit of added noise has nearly been reached in superconducting devices that take advantage of nonlinearities in Josephson junctions. Here we introduce the concept of the amplification of microwave signals using mechanical oscillation, which seems likely to enable quantum-limited operation. We drive a nanomechanical resonator with a radiation pressure force, and provide an experimental demonstration and an analytical description of how a signal input to a microwave cavity induces coherent stimulated emission and, consequently, signal amplification. This generic scheme, which is based on two linear oscillators, has the advantage of being conceptually and practically simpler than the Josephson junction devices. In our device, we achieve signal amplification of 25 decibels with the addition of 20 quanta of noise, which is consistent with the expected amount of added noise. The generality of the model allows for realization in other physical systems as well, and we anticipate that near-quantum-limited mechanical microwave amplification will soon be feasible in various applications involving integrated electrical circuits.

  14. Optical chirped beam amplification and propagation

    DOEpatents

    Barty, Christopher P.

    2004-10-12

    A short pulse laser system uses dispersive optics in a chirped-beam amplification architecture to produce high peak power pulses and high peak intensities without the potential for intensity dependent damage to downstream optical components after amplification.

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

    PubMed

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

    2014-01-01

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

  16. Sequence dependence of isothermal DNA amplification via EXPAR

    PubMed Central

    Qian, Jifeng; Ferguson, Tanya M.; Shinde, Deepali N.; Ramírez-Borrero, Alissa J.; Hintze, Arend; Adami, Christoph; Niemz, Angelika

    2012-01-01

    Isothermal nucleic acid amplification is becoming increasingly important for molecular diagnostics. Therefore, new computational tools are needed to facilitate assay design. In the isothermal EXPonential Amplification Reaction (EXPAR), template sequences with similar thermodynamic characteristics perform very differently. To understand what causes this variability, we characterized the performance of 384 template sequences, and used this data to develop two computational methods to predict EXPAR template performance based on sequence: a position weight matrix approach with support vector machine classifier, and RELIEF attribute evaluation with Naïve Bayes classification. The methods identified well and poorly performing EXPAR templates with 67–70% sensitivity and 77–80% specificity. We combined these methods into a computational tool that can accelerate new assay design by ruling out likely poor performers. Furthermore, our data suggest that variability in template performance is linked to specific sequence motifs. Cytidine, a pyrimidine base, is over-represented in certain positions of well-performing templates. Guanosine and adenosine, both purine bases, are over-represented in similar regions of poorly performing templates, frequently as GA or AG dimers. Since polymerases have a higher affinity for purine oligonucleotides, polymerase binding to GA-rich regions of a single-stranded DNA template may promote non-specific amplification in EXPAR and other nucleic acid amplification reactions. PMID:22416064

  17. Controlled Microwave Heating Accelerates Rolling Circle Amplification.

    PubMed

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

    2015-01-01

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

  18. Classroom Amplification To Enhance Student Performance.

    ERIC Educational Resources Information Center

    DiSarno, Neil J.; Schowalter, Melissa; Grassa, Patricia

    2002-01-01

    Discussion of classroom amplification systems to improve the performance of students with hearing loss or learning disabilities addresses the auditory challenges of inclusive classrooms, changing the classroom environment to reduce noise, types of amplification systems, and what teachers observe about amplification. (Contains references.) (DB)

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

    PubMed

    Miska, Eric A; Ferguson-Smith, Anne C

    2016-10-07

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

  20. Frequency domain optical parametric amplification

    PubMed Central

    Schmidt, Bruno E.; Thiré, Nicolas; Boivin, Maxime; Laramée, Antoine; Poitras, François; Lebrun, Guy; Ozaki, Tsuneyuki; Ibrahim, Heide; Légaré, François

    2014-01-01

    Today’s ultrafast lasers operate at the physical limits of optical materials to reach extreme performances. Amplification of single-cycle laser pulses with their corresponding octave-spanning spectra still remains a formidable challenge since the universal dilemma of gain narrowing sets limits for both real level pumped amplifiers as well as parametric amplifiers. We demonstrate that employing parametric amplification in the frequency domain rather than in time domain opens up new design opportunities for ultrafast laser science, with the potential to generate single-cycle multi-terawatt pulses. Fundamental restrictions arising from phase mismatch and damage threshold of nonlinear laser crystals are not only circumvented but also exploited to produce a synergy between increased seed spectrum and increased pump energy. This concept was successfully demonstrated by generating carrier envelope phase stable, 1.43 mJ two-cycle pulses at 1.8 μm wavelength. PMID:24805968

  1. Hybrid chirped pulse amplification system

    SciTech Connect

    Barty, Christopher P.; Jovanovic, Igor

    2005-03-29

    A hybrid chirped pulse amplification system wherein a short-pulse oscillator generates an oscillator pulse. The oscillator pulse is stretched to produce a stretched oscillator seed pulse. A pump laser generates a pump laser pulse. The stretched oscillator seed pulse and the pump laser pulse are directed into an optical parametric amplifier producing an optical parametric amplifier output amplified signal pulse and an optical parametric amplifier output unconverted pump pulse. The optical parametric amplifier output amplified signal pulse and the optical parametric amplifier output laser pulse are directed into a laser amplifier producing a laser amplifier output pulse. The laser amplifier output pulse is compressed to produce a recompressed hybrid chirped pulse amplification pulse.

  2. Deterministic noiseless amplification of coherent states

    NASA Astrophysics Data System (ADS)

    Hu, Meng-Jun; Zhang, Yong-Sheng

    2015-08-01

    A universal deterministic noiseless quantum amplifier has been shown to be impossible. However, probabilistic noiseless amplification of a certain set of states is physically permissible. Regarding quantum state amplification as quantum state transformation, we show that deterministic noiseless amplification of coherent states chosen from a proper set is attainable. The relation between input coherent states and gain of amplification for deterministic noiseless amplification is thus derived. Furthermore, we extend our result to more general situation and show that deterministic noiseless amplification of Gaussian states is also possible. As an example of application, we find that our amplification model can obtain better performance in homodyne detection to measure the phase of state selected from a certain set. Besides, other possible applications are also discussed.

  3. Rolling circle amplification detection of RNA and DNA

    DOEpatents

    Christian, Allen T.; Pattee, Melissa S.; Attix, Cristina M.; Tucker, James D.

    2004-08-31

    Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

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

    ERIC Educational Resources Information Center

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

    2010-01-01

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

  5. The Promise and Pitfalls of Sequence-Based Identification of Plant Pathogenic Fungi and Oomycetes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sequences of selected marker loci have been widely used for the identification of specific pathogens and the development of sequence-based diagnostic methods. Although such approaches offer several advantages over traditional culture-based methods for pathogen diagnosis and identification, they have...

  6. Predicting Salmonella enterica subsp. enterica Serotypes by Repetitive Extragenic Palindromic Sequence-Based PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DiversiLabTM System, which employs repetitive extragenic palindromic sequence-based PCR (rep-PCR) to genotype microorganisms, was evaluated as a method to predict the serotype of Salmonella isolates. Two hundred and thirty-three Salmonella isolates belonging to 14 frequently isolated serotypes f...

  7. Capture and Direct Amplification of DNA on Chitosan Microparticles in a Single PCR-Optimal Solution.

    PubMed

    Pandit, Kunal R; Nanayakkara, Imaly A; Cao, Weidong; Raghavan, Srinivasa R; White, Ian M

    2015-11-03

    While nucleic acid amplification tests have great potential as tools for rapid diagnostics, complicated sample preparation requirements inhibit their use in near-patient diagnostics and low-resource-setting applications. Recent advancements in nucleic acid purification have leveraged pH-modulated charge switching polymers to reduce the number of steps required for sample preparation. The polycation chitosan (pKa 6.4) has been used to efficiently purify DNA by binding nucleic acids in acidic buffers and then eluting them at a pH higher than 8.0. Though it is an improvement over conventional methods, this multistep procedure has not transformed the application of nucleic acid amplification assays. Here we describe a simpler approach using magnetic chitosan microparticles that interact with DNA in a manner that has not been reported before. The microparticles capture DNA at a pH optimal for PCR (8.5) just as efficiently as at low pH. Importantly, the captured DNA is still accessible by polymerase, enabling direct amplification from the microparticles. We demonstrate quantitative PCR from DNA captured on the microparticles, thus eliminating nearly all of the sample preparation steps. We anticipate that this new streamlined method for preparing DNA for amplification will greatly expand the diagnostic applications of nucleic acid amplification tests.

  8. Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

    PubMed

    Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

    2017-03-02

    Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10(-15) M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

  9. Microgel Tethering For Microarray-Based Nucleic Acid Diagnostics

    NASA Astrophysics Data System (ADS)

    Dai, Xiaoguang

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

  10. Chemical Amplification with Encapsulated Reagents

    NASA Technical Reports Server (NTRS)

    Chen, Jian; Koemer, Steffi; Craig, Stephen; Lin, Shirley; Rudkevich, Dmitry M.; Rebek, Julius, Jr.

    2002-01-01

    Autocatalysis and chemical amplification are characteristic properties of living systems, and they give rise to behaviors such as increased sensitivity, responsiveness, and self-replication. Here we report a synthetic system in which a unique form of compartmentalization leads to nonlinear, autocatalytic behavior. The compartment is a reversibly formed capsule in which a reagent is sequestered. Reaction products displace the reagent from the capsule into solution and the reaction rate is accelerated. The resulting self-regulation is sensitive to the highly selective molecular recognition properties of the capsule.

  11. Ultrasmall volume molecular isothermal amplification in microfluidic chip with advanced surface processing

    NASA Astrophysics Data System (ADS)

    Huang, Guoliang; Ma, Li; Yang, Xiaoyong; Yang, Xu

    2011-01-01

    In this paper, we developed a metal micro-fluidic chip with advanced surface processing for ultra-small volume molecular isothermal amplification. This method takes advantages of the nucleic acid amplification with good stability and consistency, high sensitivity about 31 genomic DNA copies and bacteria specific gene identification. Based on the advanced surface processing, the bioreaction assays of nucleic acid amplification was dropped about 392nl in volume. A high numerical aperture confocal optical detection system was advanced to sensitively monitor the DNA amplification with low noise and high power collecting fluorescence near to the optical diffraction limit. A speedy nucleic acid isothermal amplification was performed in the ultra-small volume microfluidic chip, where the time at the inflexions of second derivative to DNA exponential amplified curves was brought forward and the sensitivity was improved about 65 folds to that of in current 25μl Ep-tube amplified reaction, which indicates a promising clinic molecular diagnostics in the droplet amplification.

  12. EASE-MM: Sequence-Based Prediction of Mutation-Induced Stability Changes with Feature-Based Multiple Models.

    PubMed

    Folkman, Lukas; Stantic, Bela; Sattar, Abdul; Zhou, Yaoqi

    2016-03-27

    Protein engineering and characterisation of non-synonymous single nucleotide variants (SNVs) require accurate prediction of protein stability changes (ΔΔGu) induced by single amino acid substitutions. Here, we have developed a new prediction method called Evolutionary, Amino acid, and Structural Encodings with Multiple Models (EASE-MM), which comprises five specialised support vector machine (SVM) models and makes the final prediction from a consensus of two models selected based on the predicted secondary structure and accessible surface area of the mutated residue. The new method is applicable to single-domain monomeric proteins and can predict ΔΔGu with a protein sequence and mutation as the only inputs. EASE-MM yielded a Pearson correlation coefficient of 0.53-0.59 in 10-fold cross-validation and independent testing and was able to outperform other sequence-based methods. When compared to structure-based energy functions, EASE-MM achieved a comparable or better performance. The application to a large dataset of human germline non-synonymous SNVs showed that the disease-causing variants tend to be associated with larger magnitudes of ΔΔGu predicted with EASE-MM. The EASE-MM web-server is available at http://sparks-lab.org/server/ease.

  13. An empirical study on the matrix-based protein representations and their combination with sequence-based approaches.

    PubMed

    Nanni, Loris; Lumini, Alessandra; Brahnam, Sheryl

    2013-03-01

    Many domains have a stake in the development of reliable systems for automatic protein classification. Of particular interest in recent studies of automatic protein classification is the exploration of new methods for extracting features from a protein that enhance classification for specific problems. These methods have proven very useful in one or two domains, but they have failed to generalize well across several domains (i.e. classification problems). In this paper, we evaluate several feature extraction approaches for representing proteins with the aim of sequence-based protein classification. Several protein representations are evaluated, those starting from: the position specific scoring matrix (PSSM) of the proteins; the amino-acid sequence; a matrix representation of the protein, of dimension (length of the protein) ×20, obtained using the substitution matrices for representing each amino-acid as a vector. A valuable result is that a texture descriptor can be extracted from the PSSM protein representation which improves the performance of standard descriptors based on the PSSM representation. Experimentally, we develop our systems by comparing several protein descriptors on nine different datasets. Each descriptor is used to train a support vector machine (SVM) or an ensemble of SVM. Although different stand-alone descriptors work well on some datasets (but not on others), we have discovered that fusion among classifiers trained using different descriptors obtains a good performance across all the tested datasets. Matlab code/Datasets used in the proposed paper are available at http://www.bias.csr.unibo.it\

  14. Trophic amplification of climate warming.

    PubMed

    Kirby, Richard R; Beaugrand, Gregory

    2009-12-07

    Ecosystems can alternate suddenly between contrasting persistent states due to internal processes or external drivers. It is important to understand the mechanisms by which these shifts occur, especially in exploited ecosystems. There have been several abrupt marine ecosystem shifts attributed either to fishing, recent climate change or a combination of these two drivers. We show that temperature has been an important driver of the trophodynamics of the North Sea, a heavily fished marine ecosystem, for nearly 50 years and that a recent pronounced change in temperature established a new ecosystem dynamic regime through a series of internal mechanisms. Using an end-to-end ecosystem approach that included primary producers, primary, secondary and tertiary consumers, and detritivores, we found that temperature modified the relationships among species through nonlinearities in the ecosystem involving ecological thresholds and trophic amplifications. Trophic amplification provides an alternative mechanism to positive feedback to drive an ecosystem towards a new dynamic regime, which in this case favours jellyfish in the plankton and decapods and detritivores in the benthos. Although overfishing is often held responsible for marine ecosystem degeneration, temperature can clearly bring about similar effects. Our results are relevant to ecosystem-based fisheries management (EBFM), seen as the way forward to manage exploited marine ecosystems.

  15. Trophic amplification of climate warming

    PubMed Central

    Kirby, Richard R.; Beaugrand, Gregory

    2009-01-01

    Ecosystems can alternate suddenly between contrasting persistent states due to internal processes or external drivers. It is important to understand the mechanisms by which these shifts occur, especially in exploited ecosystems. There have been several abrupt marine ecosystem shifts attributed either to fishing, recent climate change or a combination of these two drivers. We show that temperature has been an important driver of the trophodynamics of the North Sea, a heavily fished marine ecosystem, for nearly 50 years and that a recent pronounced change in temperature established a new ecosystem dynamic regime through a series of internal mechanisms. Using an end-to-end ecosystem approach that included primary producers, primary, secondary and tertiary consumers, and detritivores, we found that temperature modified the relationships among species through nonlinearities in the ecosystem involving ecological thresholds and trophic amplifications. Trophic amplification provides an alternative mechanism to positive feedback to drive an ecosystem towards a new dynamic regime, which in this case favours jellyfish in the plankton and decapods and detritivores in the benthos. Although overfishing is often held responsible for marine ecosystem degeneration, temperature can clearly bring about similar effects. Our results are relevant to ecosystem-based fisheries management (EBFM), seen as the way forward to manage exploited marine ecosystems. PMID:19740882

  16. Dynamics and control of DNA sequence amplification

    NASA Astrophysics Data System (ADS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-10-01

    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.

  17. Dynamics and control of DNA sequence amplification

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-10-28

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

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

    PubMed Central

    2010-01-01

    Sequence based T-cell epitope predictions have improved immensely in the last decade. From predictions of peptide binding to major histocompatibility complex molecules with moderate accuracy, limited allele coverage, and no good estimates of the other events in the antigen-processing pathway, the field has evolved significantly. Methods have now been developed that produce highly accurate binding predictions for many alleles and integrate both proteasomal cleavage and transport events. Moreover have so-called pan-specific methods been developed, which allow for prediction of peptide binding 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 epitopes. We will focus on some of the most accurate methods and their basic background. PMID:21067545

  19. Sequencing-based breast cancer diagnostics as an alternative to routine biomarkers

    PubMed Central

    Rantalainen, Mattias; Klevebring, Daniel; Lindberg, Johan; Ivansson, Emma; Rosin, Gustaf; Kis, Lorand; Celebioglu, Fuat; Fredriksson, Irma; Czene, Kamila; Frisell, Jan; Hartman, Johan; Bergh, Jonas; Grönberg, Henrik

    2016-01-01

    Sequencing-based breast cancer diagnostics have the potential to replace routine biomarkers and provide molecular characterization that enable personalized precision medicine. Here we investigate the concordance between sequencing-based and routine diagnostic biomarkers and to what extent tumor sequencing contributes clinically actionable information. We applied DNA- and RNA-sequencing to characterize tumors from 307 breast cancer patients with replication in up to 739 patients. We developed models to predict status of routine biomarkers (ER, HER2,Ki-67, histological grade) from sequencing data. Non-routine biomarkers, including mutations in BRCA1, BRCA2 and ERBB2(HER2), and additional clinically actionable somatic alterations were also investigated. Concordance with routine diagnostic biomarkers was high for ER status (AUC = 0.95;AUC(replication) = 0.97) and HER2 status (AUC = 0.97;AUC(replication) = 0.92). The transcriptomic grade model enabled classification of histological grade 1 and histological grade 3 tumors with high accuracy (AUC = 0.98;AUC(replication) = 0.94). Clinically actionable mutations in BRCA1, BRCA2 and ERBB2(HER2) were detected in 5.5% of patients, while 53% had genomic alterations matching ongoing or concluded breast cancer studies. Sequencing-based molecular profiling can be applied as an alternative to histopathology to determine ER and HER2 status, in addition to providing improved tumor grading and clinically actionable mutations and molecular subtypes. Our results suggest that sequencing-based breast cancer diagnostics in a near future can replace routine biomarkers. PMID:27901097

  20. Microbial Contamination in Next Generation Sequencing: Implications for Sequence-Based Analysis of Clinical Samples

    PubMed Central

    Strong, Michael J.; Xu, Guorong; Morici, Lisa; Splinter Bon-Durant, Sandra; Baddoo, Melody; Lin, Zhen; Fewell, Claire; Taylor, Christopher M.; Flemington, Erik K.

    2014-01-01

    The high level of accuracy and sensitivity of next generation sequencing for quantifying genetic material across organismal boundaries gives it tremendous potential for pathogen discovery and diagnosis in human disease. Despite this promise, substantial bacterial contamination is routinely found in existing human-derived RNA-seq datasets that likely arises from environmental sources. This raises the need for stringent sequencing and analysis protocols for studies investigating sequence-based microbial signatures in clinical samples. PMID:25412476

  1. Chirality Amplification in Tactoids of Lyotropic Chromonic Liquid Crystals

    NASA Astrophysics Data System (ADS)

    Peng, Chenhui; Lavrentovich, Oleg

    2014-03-01

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

  2. Structure- and Sequence-Based Function Prediction for Non-Homologous Proteins

    PubMed Central

    Sael, Lee; Chitale, Meghana; Kihara, Daisuke

    2012-01-01

    The structural genomics projects have been accumulating an increasing number of protein structures, many of which remain functionally unknown. In parallel effort to experimental methods, computational methods are expected to make a significant contribution for functional elucidation of such proteins. However, conventional computational methods that transfer functions from homologous proteins do not help much for these uncharacterized protein structures because they do not have apparent structural or sequence similarity with the known proteins. Here, we briefly review two avenues of computational function prediction methods, i.e. structure-based methods and sequence-based methods. The focus is on our recently developments of local structure-based methods and sequence-based methods, which can effectively extract function information from distantly related proteins. Two structure-based methods, Pocket-Surfer and Patch-Surfer, identify similar known ligand binding sites for pocket regions in a query protein without using global protein fold similarity information. Two sequence-based methods, PFP and ESG, make use of weakly similar sequences that are conventionally discarded in homology based function annotation. Combined together with experimental methods we hope that computational methods will make leading contribution in functional elucidation of the protein structures. PMID:22270458

  3. [Principle of LAMP method--a simple and rapid gene amplification method].

    PubMed

    Ushikubo, Hiroshi

    2004-06-01

    So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

  4. Multiscale image contrast amplification (MUSICA)

    NASA Astrophysics Data System (ADS)

    Vuylsteke, Pieter; Schoeters, Emile P.

    1994-05-01

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

  5. Third Sound Amplification and Detailed Balance

    SciTech Connect

    Eddinger, J. D.; Ellis, F. M.

    2006-09-07

    Condensation of atoms from the vapor into a third sound resonance is expected to be capable of acoustic amplification. This results from normal to superfluid conversion that coherently accommodates atoms into the third sound velocity field. Consideration of third sound in light of the equilibrium detailed balance between vapor particles and the superfluid film provides further evidence that acoustic amplification is attainable.

  6. Classroom Amplification Technology: Theory and Practice.

    ERIC Educational Resources Information Center

    Crandell, Carl C.; Smaldino, Joseph J.

    2000-01-01

    This article reviews some relevant events in the development of acoustical standards for classrooms, describes classroom challenges to providing clear acoustical signals to children in classrooms, and outlines amplification solutions to some of those classroom challenges. Solutions include personal amplification devices and use of signal-to-noise…

  7. Short communication: characterization of DRB3 alleles in the MHC of Japanese shorthorn cattle by polymerase chain reaction-sequence-based typing.

    PubMed

    Takeshima, S; Nakai, Y; Ohta, M; Aida, Y

    2002-06-01

    A study was made of exon 2 of the bovine leukocyte antigen BoLA-DRB3 gene of 176 Japanese Shorthorn cattle at six farms in Japan using polymerase chain reaction-sequence-based typing (PCR-SBT). An initial round of PCR using conserved locus-specific primers, a second round of PCR using a locus-specific primer, and at least one sequence-specific primer (SSP), followed by direct sequencing of products of PCR with SSP were conducted. Twenty-one BoLA-DRB3 alleles were identified with frequencies ranging from 0.3 to 19.6% in 176 individuals, and two of these alleles were new alleles that have not been reported previously. The three most frequently observed alleles (DRB3*1201, *0301, and *0801) accounted for 43.8% of the alleles in the population of these herds. Next, we tested the products of amplification by PCR of BoLA-DRB3 exon 2 with RsaI, BstYI, and HaeIII, and identified 18 previously described PCR-restriction fragment length polymorphism (RFLP) types. The PCR-RFLP types reflected the results of PCR-SBT exactly. Our results indicate that exon 2 of the BoLA-DRB3 gene is highly polymorphic in Japanese Shorthorn cattle.

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

    PubMed

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

    2014-07-01

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

  9. Preferential Amplification of Pathogenic Sequences.

    PubMed

    Ge, Fang; Parker, Jayme; Chul Choi, Sang; Layer, Mark; Ross, Katherine; Jilly, Bernard; Chen, Jack

    2015-06-11

    The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called "Preferential Amplification of Pathogenic Sequences (PATHseq)" that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called "non-human primers" that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

  10. Toughness amplification in natural composites

    NASA Astrophysics Data System (ADS)

    Barthelat, Francois; Rabiei, Reza

    2011-04-01

    Natural structural materials such as bone and seashells are made of relatively weak building blocks, yet they exhibit remarkable combinations of stiffness, strength and toughness. This performance can be largely explained by their "staggered microstructure": stiff inclusions of high aspect ratio are laid parallel to each other with some overlap, and bonded by a softer matrix. While stiffness and strength are now well understood for staggered composites, the mechanisms involved in fracture are still largely unknown. This is a significant lack since the amplification of toughness with respect to their components is by far the most impressive feature in natural staggered composites such as nacre or bone. Here a model capturing the salient mechanisms involved in the cracking of a staggered structure is presented. We show that the pullout of inclusions and large process zones lead to tremendous toughness by far exceeding that of individual components. The model also suggests that a material like nacre cannot reach steady state cracking, with the implication that the toughness increases indefinitely with crack advance. These findings agree well with existing fracture data, and for the first time relate microstructural parameters with overall toughness. These insights will prove useful in the design of biomimetic materials, and provide clues on how bone fractures at the nano and microscales.

  11. SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data.

    PubMed

    Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R; Krogh, Anders; Vinther, Jeppe

    2015-05-01

    Selective 2' Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA-RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing.

  12. SHAPE Selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data

    PubMed Central

    Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R.; Krogh, Anders; Vinther, Jeppe

    2015-01-01

    Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is an accurate method for probing of RNA secondary structure. In existing SHAPE methods, the SHAPE probing signal is normalized to a no-reagent control to correct for the background caused by premature termination of the reverse transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES-based selection of cDNA–RNA hybrids on streptavidin beads effectively removes the large majority of background signal present in SHAPE probing data and that sequencing-based SHAPES data contain the same amount of RNA structure data as regular sequencing-based SHAPE data obtained through normalization to a no-reagent control. Moreover, the selection efficiently enriches for probed RNAs, suggesting that the SHAPES strategy will be useful for applications with high-background and low-probing signal such as in vivo RNA structure probing. PMID:25805860

  13. The Spatial Pattern of Cochlear Amplification

    PubMed Central

    Fisher, Jonathan A.N.; Nin, Fumiaki; Reichenbach, Tobias; Uthaiah, Revathy C.; Hudspeth, A.J.

    2012-01-01

    SUMMARY Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces. PMID:23217746

  14. Earthquake ground motion amplification for surface waves

    NASA Astrophysics Data System (ADS)

    Bowden, Daniel C.; Tsai, Victor C.

    2017-01-01

    Surface waves from earthquakes are known to cause strong damage, especially for larger structures such as skyscrapers and bridges. However, common practice in characterizing seismic hazard at a specific site considers the effect of near-surface geology on only vertically propagating body waves. Here we show that surface waves have a unique and different frequency-dependent response to known geologic structure and that this amplification can be analytically calculated in a manner similar to current hazard practices. Applying this framework to amplification in the Los Angeles Basin, we find that peak ground accelerations for certain large regional earthquakes are underpredicted if surface waves are not properly accounted for and that the frequency of strongest ground motion amplification can be significantly different. Including surface-wave amplification in hazards calculations is therefore essential for accurate predictions of strong ground motion for future San Andreas Fault ruptures.

  15. A Simple Structure for Signal Amplification

    NASA Astrophysics Data System (ADS)

    Ding, Wan-Xiang; Gu, Chang-Gui; Liang, Xiao-Ming

    2016-02-01

    It has been found that a triple-node feed-forward motif has a function of signal amplification, where two input nodes receive the external weak signal and jointly modulate the response of the third output node [Liang et al., Phys. Rev. E 88 (2013) 012910]. We here show that the signal amplification can be further enhanced by adding a link between the two input nodes in the feed-forward motif. We further reveal that the coupling strength of the link regulates the enhancement of signal amplification in the modified feed-forward motif. We finally analyze the mechanism of signal amplification of such simple structure. Supported by the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning under Grant No. QD2015016, the National Natural Science Foundation of China under Grant Nos. 11505114 and 11305078

  16. AMPLIFICATION OF RIBOSOMAL RNA SEQUENCES - Book Chapter

    EPA Science Inventory

    This book chapter contains the following headings and subheadings: Introduction; Experimental Approach - Precautions, Template, Primers, Reaction Conditions, Enhancers, Post Amplification; Procedures - Template DNA, Basic PCR, Thermal Cycle Parameters, Enzyme Addition, Agarose Ge...

  17. Coupled isothermal polynucleotide amplification and translation system

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor)

    1998-01-01

    A cell-free system for polynucleotide amplification and translation is disclosed. Also disclosed are methods for using the system and a composition which allows the various components of the system to function under a common set of reaction conditions.

  18. The spatial pattern of cochlear amplification.

    PubMed

    Fisher, Jonathan A N; Nin, Fumiaki; Reichenbach, Tobias; Uthaiah, Revathy C; Hudspeth, A J

    2012-12-06

    Sensorineural hearing loss, which stems primarily from the failure of mechanosensory hair cells, changes the traveling waves that transmit acoustic signals along the cochlea. However, the connection between cochlear mechanics and the amplificatory function of hair cells remains unclear. Using an optical technique that permits the targeted inactivation of prestin, a protein of outer hair cells that generates forces on the basilar membrane, we demonstrate that these forces interact locally with cochlear traveling waves to achieve enormous mechanical amplification. By perturbing amplification in narrow segments of the basilar membrane, we further show that a cochlear traveling wave accumulates gain as it approaches its peak. Analysis of these results indicates that cochlear amplification produces negative damping that counters the viscous drag impeding traveling waves; targeted photoinactivation locally interrupts this compensation. These results reveal the locus of amplification in cochlear traveling waves and connect the characteristics of normal hearing to molecular forces.

  19. Rolling circle amplification of metazoan mitochondrialgenomes

    SciTech Connect

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

    2005-07-31

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

  20. Amplification of chromosomal DNA in situ

    DOEpatents

    Christian, Allen T.; Coleman, Matthew A.; Tucker, James D.

    2002-01-01

    Amplification of chromosomal DNA in situ to increase the amount of DNA associated with a chromosome or chromosome region is described. The amplification of chromosomal DNA in situ provides for the synthesis of Fluorescence in situ Hybridization (FISH) painting probes from single dissected chromosome fragments, the production of cDNA libraries from low copy mRNAs and improved in Comparative Genomic Hybridization (CGH) procedures.

  1. Amplification uncertainty relation for probabilistic amplifiers

    NASA Astrophysics Data System (ADS)

    Namiki, Ryo

    2015-09-01

    Traditionally, quantum amplification limit refers to the property of inevitable noise addition on canonical variables when the field amplitude of an unknown state is linearly transformed through a quantum channel. Recent theoretical studies have determined amplification limits for cases of probabilistic quantum channels or general quantum operations by specifying a set of input states or a state ensemble. However, it remains open how much excess noise on canonical variables is unavoidable and whether there exists a fundamental trade-off relation between the canonical pair in a general amplification process. In this paper we present an uncertainty-product form of amplification limits for general quantum operations by assuming an input ensemble of Gaussian-distributed coherent states. It can be derived as a straightforward consequence of canonical uncertainty relations and retrieves basic properties of the traditional amplification limit. In addition, our amplification limit turns out to give a physical limitation on probabilistic reduction of an Einstein-Podolsky-Rosen uncertainty. In this regard, we find a condition that probabilistic amplifiers can be regarded as local filtering operations to distill entanglement. This condition establishes a clear benchmark to verify an advantage of non-Gaussian operations beyond Gaussian operations with a feasible input set of coherent states and standard homodyne measurements.

  2. Onshore seismic amplifications due to bathymetric features

    NASA Astrophysics Data System (ADS)

    Rodríguez-Castellanos, A.; Carbajal-Romero, M.; Flores-Guzmán, N.; Olivera-Villaseñor, E.; Kryvko, A.

    2016-08-01

    We perform numerical calculations for onshore seismic amplifications, taking into consideration the effect of bathymetric features on the propagation of seismic movements. To this end, the boundary element method is applied. Boundary elements are employed to irradiate waves and, consequently, force densities can be obtained for each boundary element. From this assumption, Huygens’ principle is applied, and since the diffracted waves are built at the boundary from which they are radiated, this idea is equivalent to Somigliana’s representation theorem. The application of boundary conditions leads to a linear system being obtained (Fredholm integral equations). Several numerical models are analyzed, with the first one being used to verify the proposed formulation, and the others being used to estimate onshore seismic amplifications due to the presence of bathymetric features. The results obtained show that compressional waves (P-waves) generate onshore seismic amplifications that can vary from 1.2 to 5.2 times the amplitude of the incident wave. On the other hand, the shear waves (S-waves) can cause seismic amplifications of up to 4.0 times the incident wave. Furthermore, an important result is that in most cases the highest seismic amplifications from an offshore earthquake are located on the shoreline and not offshore, despite the seafloor configuration. Moreover, the influence of the incident angle of seismic waves on the seismic amplifications is highlighted.

  3. The Discovery of Rolling Circle Amplification and Rolling Circle Transcription.

    PubMed

    Mohsen, Michael G; Kool, Eric T

    2016-11-15

    Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently

  4. A Rapid and Simple Integrated Extraction Amplification and Detection Device for Y. pestis

    DTIC Science & Technology

    2000-10-01

    strumented technologies of DNA microarrays and related - microfluidics . 3- 5 By contrast, our company is focusing on the development of relatively simple... radioactive labels into the Ed.. Cold Spring Harbor Laboratory. Cold Spring Harbor. amplification reactions for the detection of nucleic acids is N.Y

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

    PubMed Central

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

    2014-01-01

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

  6. Heat induces gene amplification in cancer cells

    SciTech Connect

    Yan, Bin; Ouyang, Ruoyun; Huang, Chenghui; Liu, Franklin; Neill, Daniel; Li, Chuanyuan; Dewhirst, Mark

    2012-10-26

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

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

    PubMed Central

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

    2010-01-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods—traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit—showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population. PMID:20392917

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

    PubMed

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

    2010-06-01

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

  9. Emerging pathogens in the fish farming industry and sequencing-based pathogen discovery.

    PubMed

    Tengs, Torstein; Rimstad, Espen

    2017-02-03

    The use of large scale DNA/RNA sequencing has become an integral part of biomedical research. Reduced sequencing costs and the availability of efficient computational resources has led to a revolution in how problems concerning genomics and transcriptomics are addressed. Sequencing-based pathogen discovery represents one example of how genetic data can now be used in ways that were previously considered infeasible. Emerging pathogens affect both human and animal health due to a multitude of factors, including globalization, a shifting environment and an increasing human population. Fish farming represents a relevant, interesting and challenging system to study emerging pathogens. This review summarizes recent progress in pathogen discovery using sequence data, with particular emphasis on viruses in Atlantic salmon (Salmo salar).

  10. The promise and pitfalls of sequence-based identification of plant-pathogenic fungi and oomycetes.

    PubMed

    Kang, Seogchan; Mansfield, Michele A; Park, Bongsoo; Geiser, David M; Ivors, Kelly L; Coffey, Michael D; Grünwald, Niklaus J; Martin, Frank N; Lévesque, C André; Blair, Jaime E

    2010-08-01

    Sequences of selected marker loci have been widely used for the identification of specific pathogens and the development of sequence-based diagnostic methods. Although such approaches offer several advantages over traditional culture-based methods for pathogen diagnosis and identification, they have their own pitfalls. These include erroneous and incomplete data in reference databases, poor or oversimplified interpretation of search results, and problems associated with defining species boundaries. In this letter, we outline the potential benefits and drawbacks of using sequence data for identification and taxonomic deduction of plant-pathogenic fungi and oomycetes, using phytophthora as a primary example. We also discuss potential remedies for these pitfalls and address why coordinated community efforts are essential to make such remedies more efficient and robust.

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

    NASA Astrophysics Data System (ADS)

    Zhang, Chongfu; Qiu, Kun; Ma, Chunli

    2009-11-01

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

  12. Single-cell sequencing-based technologies will revolutionize whole-organism science.

    PubMed

    Shapiro, Ehud; Biezuner, Tamir; Linnarsson, Sten

    2013-09-01

    The unabated progress in next-generation sequencing technologies is fostering a wave of new genomics, epigenomics, transcriptomics and proteomics technologies. These sequencing-based technologies are increasingly being targeted to individual cells, which will allow many new and longstanding questions to be addressed. For example, single-cell genomics will help to uncover cell lineage relationships; single-cell transcriptomics will supplant the coarse notion of marker-based cell types; and single-cell epigenomics and proteomics will allow the functional states of individual cells to be analysed. These technologies will become integrated within a decade or so, enabling high-throughput, multi-dimensional analyses of individual cells that will produce detailed knowledge of the cell lineage trees of higher organisms, including humans. Such studies will have important implications for both basic biological research and medicine.

  13. Capture and Amplification by Tailing and Switching (CATS)

    PubMed Central

    Turchinovich, Andrey; Surowy, Harald; Serva, Andrius; Zapatka, Marc; Lichter, Peter; Burwinkel, Barbara

    2014-01-01

    Massive parallel sequencing (MPS) technologies have paved the way into new areas of research including individualized medicine. However, sequencing of trace amounts of DNA or RNA still remains a major challenge, especially for degraded nucleic acids like circulating DNA. This together with high cost and time requirements impedes many important applications of MPS in medicine and fundamental science. We have established a fast, cheap and highly efficient protocol called ‘Capture and Amplification by Tailing and Switching’ (CATS) to directly generate ready-to-sequence libraries for MPS from nanogram and picogram quantities of both DNA and RNA. Furthermore, those DNA libraries are strand-specific, can be prepared within 2–3 h and do not require preliminary sample amplification steps. To exemplify the capacity of the technique, we have generated and sequenced DNA libraries from hundred-picogram amounts of circulating nucleic acids isolated from human blood plasma, one nanogram of mRNA-enriched total RNA from cultured cells and few nanograms of bisulfite-converted DNA. The approach for DNA library preparation from minimal and fragmented input described here will find broad application in diverse research areas such as translational medicine including therapy monitoring, prediction, prognosis and early detection of various human disorders and will permit high-throughput DNA sequencing from previously inaccessible material such as minute forensic and archeological samples. PMID:24922482

  14. Advances in nucleic acid-based detection methods.

    PubMed Central

    Wolcott, M J

    1992-01-01

    Laboratory techniques based on nucleic acid methods have increased in popularity over the last decade with clinical microbiologists and other laboratory scientists who are concerned with the diagnosis of infectious agents. This increase in popularity is a result primarily of advances made in nucleic acid amplification and detection techniques. Polymerase chain reaction, the original nucleic acid amplification technique, changed the way many people viewed and used nucleic acid techniques in clinical settings. After the potential of polymerase chain reaction became apparent, other methods of nucleic acid amplification and detection were developed. These alternative nucleic acid amplification methods may become serious contenders for application to routine laboratory analyses. This review presents some background information on nucleic acid analyses that might be used in clinical and anatomical laboratories and describes some recent advances in the amplification and detection of nucleic acids. PMID:1423216

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

    PubMed

    Boyle, David S; McNerney, Ruth; Teng Low, Hwee; Leader, Brandon Troy; Pérez-Osorio, Ailyn C; Meyer, Jessica C; O'Sullivan, Denise M; Brooks, David G; Piepenburg, Olaf; Forrest, Matthew S

    2014-01-01

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

  16. A novel HLA-B allele, B*58:01:12, detected in a Taiwanese volunteer bone marrow stem cell donor using sequence-based typing method.

    PubMed

    Yang, K L; Lee, S K; Kao, R H; Lin, C L; Lin, P Y

    2013-08-01

    Human leukocyte antigen-B*58:01:12, a novel rare allele of HLA-B*58:01 variant, was found in a Taiwanese volunteer bone marrow donor by SBT (sequence-based typing) method. The DNA sequence of B*58:01:12 is identical to the sequence of B*58:01:01 in exons 2, 3 and 4 except at nucleotide position 483 where nucleotide C is substituted by T (at codon 137; GAC GAT). Due to the silent point mutation, the amino acid sequence of B*58:01:12 is identical to the sequence of B*58:01:01. The HLA haplotype in association with B*58:01:12 may be deduced as A*33:03-B*58:01:12-DRB1*03:01. The discovery of B*58:01:12 adds further polymorphism of B*58:01 in Taiwanese population.

  17. Time varying arctic climate change amplification

    SciTech Connect

    Chylek, Petr; Dubey, Manvendra K; Lesins, Glen; Wang, Muyin

    2009-01-01

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

  18. Amplification, Redundancy, and Quantum Chernoff Information

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

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

  19. Amplification, redundancy, and quantum Chernoff information.

    PubMed

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

    2014-04-11

    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.

  20. Optical amplification enhancement in photonic crystals

    SciTech Connect

    Sapienza, R.; Leonetti, M.; Froufe-Perez, L. S.; Galisteo-Lopez, J. F.; Lopez, C.; Conti, C.

    2011-02-15

    Improving and controlling the efficiency of a gain medium is one of the most challenging problems of laser research. By measuring the gain length in an opal-based photonic crystal doped with laser dye, we demonstrate that optical amplification is more than twenty-fold enhanced along the {Gamma}-K symmetry directions of the face-centered-cubic photonic crystal. These results are theoretically explained by directional variations of the density of states, providing a quantitative connection between density of the states and light amplification.

  1. Direct loop mediated isothermal amplification on filters for quantification of Dehalobacter in groundwater.

    PubMed

    Stedtfeld, Robert D; Stedtfeld, Tiffany M; Samhan, Farag; Kanitkar, Yogendra H; Hatzinger, Paul B; Cupples, Alison M; Hashsham, Syed A

    2016-12-01

    Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 10(2)Dehalobacter cells per 100mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100mg per L of humic acid per reaction compared to 1mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter, the DFA method can potentially be used for a number of applications requiring field-able, rapid (<60min) and highly sensitive quantification of microorganisms in environmental water samples.

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

    PubMed

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

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

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

    PubMed

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

    2008-10-01

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

  4. Comprehensive evaluation of molecular enhancers of the isothermal exponential amplification reaction

    PubMed Central

    Mok, Ellie; Wee, Eugene; Wang, Yuling; Trau, Matt

    2016-01-01

    The exponential amplification reaction (EXPAR) is an emerging isothermal nucleic acid amplification method with high potential for molecular diagnostics due to its isothermal nature and high amplification efficiency. However, the use of EXPAR is limited by the high levels of non-specific amplification. Hence, methods that can improve the specificity of EXPAR are desired to facilitate its widespread adoption in practice. Herein, we proposed a strategy to improve EXPAR performance by using molecular enhancers. Eight small molecules were investigated, including ethylene glycol, propylene glycol, betaine, dimethyl sulfoxide (DMSO), trehalose, tetramethylammonium chloride (TMAC), bovine serum albumin (BSA) and single-stranded binding (SSB) proteins. A combination of kinetic and end-point analysis was adopted to investigate how these molecules affected EXPAR performance. Trehalose, TMAC, BSA and SSB proteins were found to have positive effects on EXPAR with trehalose being able to increase the efficiency of EXPAR. In contrast, TMAC, BSA and SSB proteins were shown to increase the specificity of EXPAR. We applied our findings to demonstrate the combination of trehalose and TMAC could simultaneously improve both the efficiency and specificity of an EXPAR-based miRNA detection method. The information provided in this study may serve as a reference to benefit the wider isothermal amplification community. PMID:27910874

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

    PubMed

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

    2000-01-29

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

  6. Reassessment of Sequence-Based Targets for Identification of Bacillus Species

    PubMed Central

    Blackwood, K. S.; Turenne, C. Y.; Harmsen, D.; Kabani, A. M.

    2004-01-01

    The Bacillus genus is a large heterogeneous group in need of an efficient method for species differentiation. To determine the current validity of a sequence-based method for identification and provide contemporary data, PCR and sequencing of a 500-bp product encompassing the V1 to V3 regions of the 16S rRNA gene were undertaken using 65 of the 83 type strains of this genus. This region proved discriminatory between most species (70.0 to 100% similarity), the exceptions being clinically relevant B. cereus and B. anthracis as well as nonpathogenic B. psychrotolerans and B. psychrodurans. Consequently, 27 type and clinical strains from the B. cereus group were used to test alternate targets (rpoB, vrrA, and the 16S-23S spacer region) for identification. The rpoB gene proved the best alternate target, with a conserved 4-nucleotide difference between B. cereus and B. anthracis. The high 16S rRNA gene sequence similarities between some strains demonstrated the need for a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differentiation of Medical Microorganisms mandate. Accordingly, the 16S rRNA gene sequences generated in this study have been submitted for inclusion into its publicly accessible, quality-controlled database at http://www.ridom_rdna.de/. PMID:15071016

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Tools for Sequence-Based miRNA Target Prediction: What to Choose?

    PubMed Central

    Riffo-Campos, Ángela L.; Riquelme, Ismael; Brebi-Mieville, Priscilla

    2016-01-01

    MicroRNAs (miRNAs) are defined as small non-coding RNAs ~22 nt in length. They regulate gene expression at a post-transcriptional level through complementary base pairing with the target mRNA, leading to mRNA degradation and therefore blocking translation. In the last decade, the dysfunction of miRNAs has been related to the development and progression of many diseases. Currently, researchers need a method to identify precisely the miRNA targets, prior to applying experimental approaches that allow a better functional characterization of miRNAs in biological processes and can thus predict their effects. Computational prediction tools provide a rapid method to identify putative miRNA targets. However, since a large number of tools for the prediction of miRNA:mRNA interactions have been developed, all with different algorithms, the biological researcher sometimes does not know which is the best choice for his study and many times does not understand the bioinformatic basis of these tools. This review describes the biological fundamentals of these prediction tools, characterizes the main sequence-based algorithms, and offers some insights into their uses by biologists. PMID:27941681

  9. Identifying natural substrates for chaperonins using a sequence-based approach

    PubMed Central

    Stan, George; Brooks, Bernard R.; Lorimer, George H.; Thirumalai, D.

    2005-01-01

    The Escherichia coli chaperonin machinery, GroEL, assists the folding of a number of proteins. We describe a sequence-based approach to identify the natural substrate proteins (SPs) for GroEL. Our method is based on the hypothesis that natural SPs are those that contain patterns of residues similar to those found in either GroES mobile loop and/or strongly binding peptide in complex with GroEL. The method is validated by comparing the predicted results with experimentally determined natural SPs for GroEL. We have searched for such patterns in five genomes. In the E. coli genome, we identify 1422 (about one-third) sequences that are putative natural SPs. In Saccharomyces cerevisiae, 2885 (32%) of sequences can be natural substrates for Hsp60, which is the analog of GroEL. The precise number of natural SPs is shown to be a function of the number of contacts an SP makes with the apical domain (NC) and the number of binding sites (NB) in the oligomer with which it interacts. For known SPs for GroEL, we find ~4 < NC < 5 and 2 ≤ NB ≤ 4. A limited analysis of the predicted binding sequences shows that they do not adopt any preferred secondary structure. Our method also predicts the putative binding regions in the identified SPs. The results of our study show that a variety of SPs, associated with diverse functions, can interact with GroEL. PMID:15576562

  10. Tools for Sequence-Based miRNA Target Prediction: What to Choose?

    PubMed

    Riffo-Campos, Ángela L; Riquelme, Ismael; Brebi-Mieville, Priscilla

    2016-12-09

    MicroRNAs (miRNAs) are defined as small non-coding RNAs ~22 nt in length. They regulate gene expression at a post-transcriptional level through complementary base pairing with the target mRNA, leading to mRNA degradation and therefore blocking translation. In the last decade, the dysfunction of miRNAs has been related to the development and progression of many diseases. Currently, researchers need a method to identify precisely the miRNA targets, prior to applying experimental approaches that allow a better functional characterization of miRNAs in biological processes and can thus predict their effects. Computational prediction tools provide a rapid method to identify putative miRNA targets. However, since a large number of tools for the prediction of miRNA:mRNA interactions have been developed, all with different algorithms, the biological researcher sometimes does not know which is the best choice for his study and many times does not understand the bioinformatic basis of these tools. This review describes the biological fundamentals of these prediction tools, characterizes the main sequence-based algorithms, and offers some insights into their uses by biologists.

  11. Application of Sequence-based Methods in Human MicrobialEcology

    SciTech Connect

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

    2005-08-29

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

  12. A sequence-based map of Arabidopsis genes with mutant phenotypes.

    PubMed

    Meinke, David W; Meinke, Laura K; Showalter, Thomas C; Schissel, Anna M; Mueller, Lukas A; Tzafrir, Iris

    2003-02-01

    The classical genetic map of Arabidopsis contains 462 genes with mutant phenotypes. Chromosomal locations of these genes have been determined over the past 25 years based on recombination frequencies with visible and molecular markers. The most recent update of the classical map was published in a special genome issue of Science that dealt with Arabidopsis (D.W. Meinke, J.M. Cherry, C. Dean, S.D. Rounsley, M. Koornneef [1998] Science 282: 662-682). We present here a comprehensive list and sequence-based map of 620 cloned genes with mutant phenotypes. This map documents for the first time the exact locations of large numbers of Arabidopsis genes that give a phenotype when disrupted by mutation. Such a community-based physical map should have broad applications in Arabidopsis research and should serve as a replacement for the classical genetic map in the future. Assembling a comprehensive list of genes with a loss-of-function phenotype will also focus attention on essential genes that are not functionally redundant and ultimately contribute to the identification of the minimal gene set required to make a flowering plant.

  13. Potential of a sequence-based antigenic distance measure to indicate equine influenza vaccine strain efficacy.

    PubMed

    Daly, Janet M; Elton, Debra

    2013-12-09

    The calculation of p(epitope) values, a sequence-based measure of antigenic distance between strains, was developed for human influenza. The potential to apply the p(epitope) value to equine influenza vaccine strain selection was assessed. There was a negative correlation between p(epitope) value and vaccine efficacy for pairs of vaccine and challenge strains used in cross-protection studies in ponies that just reached statistical significance (p=0.046) only if one pair of viruses was excluded from the analysis. Thus the p(epitope) value has potential to provide additional data to consider in the decision-making process for updating equine influenza vaccine strains. However, further work is required to define the epitopes of the equine H3N8 haemagglutinin protein recognised by equine antibodies, which could lead to refinement of the p(epitope) value calculation. Furthermore, other factors such as vaccine potency and virulence of circulating strains may also influence vaccine efficacy.

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

    PubMed

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

    2015-12-01

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

  15. Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein

    SciTech Connect

    Kreader, C.A.

    1996-03-01

    The benefits of adding bovine serum albumin (BSA) or T4 gene 32 proteins (gp32) to PCR were evaluated with reaction mixtures containing substances that inhibit amplification. Whereas 10- to 1,000-fold more FeCl{sub 3}, hemin, fulvic acids, humic acids, tannic acids, or extracts from feces, freshwater, or marine water were accommodated in PCR when either 400 ng of BSA per {mu}l was included in the reactions, neither BSA nor gp32 relieved interference significantly when minimum inhibitory levels of bile salts, bilirubin, EDTA, NaCl, sodium dodecyl sulfate, or Triton X-100 were present. Use of BSA and gp32 together offered no more relief of inhibition than either alone at its optimal level, and neither protein had any noticeable effect on amplification in the absence of inhibitors. 21 refs., 3 figs.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    DOEpatents

    Levitsky, Igor A.; Krivoshlykov, Sergei G.

    2004-02-03

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

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

    PubMed Central

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

    2013-01-01

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

  19. Novel DNA Polymer for Amplification Pretargeting

    PubMed Central

    2015-01-01

    In this Letter, different from conventional pretargeting, an additional novel DNA polymer with multiple copies of a target was first designed to be administrated between the antitumor antibody, and the labeled effector served as an amplification pretargeting strategy. Two phosphorothioate DNA strands, a bridging and a target strand, were hybridized to form a polymer. Polymer size, as a function of molar ratios, was then monitored by size exclusion HPLC and electrophoretic mobility shift assay. Moreover, binding efficiency of polymers with the radiolabeled effector and polymer size after hybridization were measured by HPLC as well. As the polymer was expected to produce more binding sites that would be targeted by effectors, amplification pretargeting can greatly improve accumulation of effectors in tumor. This novel proof-of-concept was then well demonstrated by the in vitro test of signal amplification in antibody-binding protein L coated plate and LS174T cells. Compared to conventional pretargeting, significantly increasing radioactive signal was observed in this designed amplification pretargeting, which would serve as a useful paradigm of the potential of oligomer polymers to improve pretargeting and other related approaches. PMID:26396682

  20. Desert Amplification in a Warming Climate

    PubMed Central

    Zhou, Liming

    2016-01-01

    Here I analyze the observed and projected surface temperature anomalies over land between 50°S-50°N for the period 1950–2099 by large-scale ecoregion and find strongest warming consistently and persistently seen over driest ecoregions such as the Sahara desert and the Arabian Peninsula during various 30-year periods, pointing to desert amplification in a warming climate. This amplification enhances linearly with the global mean greenhouse gases(GHGs) radiative forcing and is attributable primarily to a stronger GHGs-enhanced downward longwave radiation forcing reaching the surface over drier ecoregions as a consequence of a warmer and thus moister atmosphere in response to increasing GHGs. These results indicate that desert amplification may represent a fundamental pattern of global warming associated with water vapor feedbacks over land in low- and mid- latitudes where surface warming rates depend inversely on ecosystem dryness. It is likely that desert amplification might involve two types of water vapor feedbacks that maximize respectively in the tropical upper troposphere and near the surface over deserts, with both being very dry and thus extremely sensitive to changes of water vapor. PMID:27538725

  1. Nondeterministic Noiseless Linear Amplification of Quantum Systems

    NASA Astrophysics Data System (ADS)

    Ralph, T. C.; Lund, A. P.

    2009-04-01

    We introduce the concept of non-deterministic noiseless linear amplification. We propose a linear optical realization of this transformation that could be built with current technology. We discuss the application of the device to distillation of continuous variable entanglement. We demonstrate that highly pure entanglement can be distilled from transmission over a lossy channel.

  2. Detection of cochlear amplification and its activation.

    PubMed

    Dong, Wei; Olson, Elizabeth S

    2013-08-20

    The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source.

  3. Topographic amplification across a taiwanese ridge

    NASA Astrophysics Data System (ADS)

    Rault, Claire; Meunier, Patrick; Burtin, Arnaud; Marc, Odin; Weian Chao, Vvn; Wu, Yih-Min; Hovius, Niels

    2016-04-01

    A line of 6 broadband seismometers have been deployed across a ridge in the Hualien County (Eastern Taiwan) in order to study topographic amplification. Since March 2015, the network has been continuously recording waves incoming from the Taiwanese regional seismicity. The hill is well approximated by a triangular topography of 3600m in length by 900m in height. We present a preliminary analysis performed over a dozen of earthquakes selected from the Seismic Taiwanese catalog (CWBSN). We show that most of the Uphill records exhibit a systematic amplification of seismic waves (peak to peak of particle velocity) in the relevant frequency band [0.5-2Hz]. By contrast, energy within the larger frequency band [6-20Hz] reflects local site effects induced by the soil layer. We report amplification ratios ranging from ranging from 1.2 to 3 and from 1.8 to 4 for P and S waves respectively. We show that amplification processes at the top strongly depend on the parameter α defined as the angle between the azimuth of incoming wave and the azimuth of the ridge divide.

  4. Desert Amplification in a Warming Climate

    NASA Astrophysics Data System (ADS)

    Zhou, Liming

    2016-08-01

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

  5. Detection of Cochlear Amplification and Its Activation

    PubMed Central

    Dong, Wei; Olson, Elizabeth S.

    2013-01-01

    The operation of the mammalian cochlea relies on a mechanical traveling wave that is actively boosted by electromechanical forces in sensory outer hair cells (OHCs). This active cochlear amplifier produces the impressive sensitivity and frequency resolution of mammalian hearing. The cochlear amplifier has inspired scientists since its discovery in the 1970s, and is still not well understood. To explore cochlear electromechanics at the sensory cell/tissue interface, sound-evoked intracochlear pressure and extracellular voltage were measured using a recently developed dual-sensor with a microelectrode attached to a micro-pressure sensor. The resulting coincident in vivo observations of OHC electrical activity, pressure at the basilar membrane and basilar membrane displacement gave direct evidence for power amplification in the cochlea. Moreover, the results showed a phase shift of voltage relative to mechanical responses at frequencies slightly below the peak, near the onset of amplification. Based on the voltage-force relationship of isolated OHCs, the shift would give rise to effective OHC pumping forces within the traveling wave peak. Thus, the shift activates the cochlear amplifier, serving to localize and thus sharpen the frequency region of amplification. These results are the most concrete evidence for cochlear power amplification to date and support OHC somatic forces as its source. PMID:23972858

  6. Desert Amplification in a Warming Climate.

    PubMed

    Zhou, Liming

    2016-08-19

    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.

  7. Site amplifications for generic rock sites

    USGS Publications Warehouse

    Boore, D.M.; Joyner, W.B.

    1997-01-01

    Seismic shear-wave velocity as a function of depth for generic rock sites has been estimated from borehole data and studies of crustal velocities, and these velocities have been used to compute frequency-dependent amplifications for zero attenuation for use in simulations of strong ground motion. We define a generic rock site as one whose velocity at shallow depths equals the average of those from the rock sites sampled by the borehole data. Most of the boreholes are in populated areas; for that reason, the rock sites sampled are of particular engineering significance. We consider two generic rock sites: rock, corresponding to the bulk of the borehole data, and very hard rock, such as is found in glaciated regions in large areas of eastern North America or in portions of western North America. The amplifications on rock sites can be in excess of 3.5 at high frequencies, in contrast to the amplifications of less than 1.2 on very hard rock sites. The consideration of unattenuated amplification alone is computationally convenient, but what matters for ground-motion estimation is the combined effect of amplification and attenuation. For reasonable values of the attenuation parameter K0, the combined effect of attenuation and amplification for rock sites peaks between about 2 and 5 Hz with a maximum level of less than 1.8. The combined effect is about a factor of 1.5 at 1 Hz and is less than unity for frequencies in the range of 10 to 20 Hz (depending on K0). Using these amplifications, we find provisional values of about ???? = 70 bars and K0 = 0.035 sec for rock sites in western North America by fitting our empirically determined response spectra for an M 6.5 event to simulated values. The borehole data yield shear velocities (V??30) of 618 and 306 m/sec for "rock" and "soil" sites, respectively, when averaged over the upper 30 m. From this, we recommend that V??30 equals 620 and 310 m/sec for applications requiring the average velocity for rock and soil sites in

  8. Effects of polymer end groups on chemical amplification

    NASA Astrophysics Data System (ADS)

    Ito, Hiroshi; England, William P.; Lundmark, Stephen B.

    1992-06-01

    Polymer end groups could affect the sensitivity of chemical amplification resist systems based on acid catalysis in a fashion completely different from the conventional resist systems. Their acidolysis susceptibility could depend on the initiator employed in polymerization, which is illustrated by two examples in this paper. When (alpha) ,(alpha) -azobis(isobutyronitrile) is used as the radical polymerization initiator, PBOCST with a lower molecular weight provides a less sensitive tBOC resist than a higher molecular weight polymer, which is due to the poisoning effect of the CN group attached to the polymer end. Low molecular weight PBOCST's were prepared also with benzoyl peroxide and via living anionic polymerization to confirm the end group effect on the tBOC acidolysis. In contrast, there are cases where certain end groups could provide reaction sites to photochemically generated acids. One such example is poly(hydroxy-(alpha) -methylstyrene) (PHOMS). The (rho) -PHOMS prepared by heating the tBOC-protected polymer (made by cationic polymerization) undergoes extremely efficient acid-catalyzed depolymerization. In contrast, the (rho) -PHOMS made by desilylation of anionically obtained silyl-protected polymer is very inert to such acidolysis. The high sensitivity of the cationic (rho) -PHOMS is due to the presence of end groups that are introduced during accidental and/or intentional termination and that are very reactive toward acids.

  9. Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

    PubMed

    Glais, Laurent; Jacquot, Emmanuel

    2015-01-01

    Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

  10. Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

    PubMed Central

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

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the 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. PMID:25426953

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

    PubMed

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

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the 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.

  12. Rapid microfluidic thermal cycler for nucleic acid amplification

    DOEpatents

    Beer, Neil Reginald; Vafai, Kambiz

    2015-10-27

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

  13. Centrifugal Microfluidic System for Nucleic Acid Amplification and Detection

    PubMed Central

    Miao, Baogang; Peng, Niancai; Li, Lei; Li, Zheng; Hu, Fei; Zhang, Zengming; Wang, Chaohui

    2015-01-01

    We report here the development of a rapid PCR microfluidic system comprising a double-shaft turntable and centrifugal-based disc that rapidly drives the PCR mixture between chambers set at different temperatures, and the bidirectional flow improved the space utilization of the disc. Three heating resistors and thermistors maintained uniform, specific temperatures for the denaturation, annealing, and extension steps of the PCR. Infrared imaging showed that there was little thermal interference between reaction chambers; the system enabled the cycle number and reaction time of each step to be independently adjusted. To validate the function and efficiency of the centrifugal microfluidic system, a 350-base pair target gene from the hepatitis B virus was amplified and quantitated by fluorescence detection. By optimizing the cycling parameters, the reaction time was reduced to 32 min as compared to 120 min for a commercial PCR machine. DNA samples with concentrations ranging from 10 to 106 copies/mL could be quantitatively analyzed using this system. This centrifugal-based microfluidic platform is a useful system and possesses industrialization potential that can be used for portable diagnostics. PMID:26556354

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

    PubMed

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

    2005-08-01

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

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

    PubMed

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

    2016-08-01

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

  16. Frequency of HLA-A alleles in the Syrian population genotyped by sequence-based typing.

    PubMed

    Madania, A; Ghoury, I; Al-Ashkar, W; Nweder, S; Zarzour, H

    2014-10-01

    HLA-A molecules are highly polymorphic. Their accurate typing at a high-resolution level is crucial for successful organ, bone marrow and cord blood transplantation. Furthermore, several HLA alleles have been involved in susceptibility to autoimmune diseases, allergies, cancers and inflammations. In order to determine common HLA-A alleles in Syria and their frequencies, sequence-based typing (SBT) was used to genotype HLA-A alleles at high resolution (four digit level) among one hundred and thirty randomly selected Syrian individuals. Exons 2, 3 and 4 of the HLA-A gene were amplified by PCR and sequenced. The sbt-engine software was used for allele assignment. Ambiguities were solved using group-specific sequencing primers (GSSPs). We could identify 32 different HLA-A alleles which were divided into 3 groups: high frequency (approximately 10%, A*01:01; A*24:02; A*03:01; A*02:01), moderate frequency (approximately 3%, such as A*02:05, A*31:01 and A*33:01), and low frequency (approximately 1%, such as A*02:11, A*29:01, A*02:02 and A*36:01). Homozygosity rate was higher than expected (11.5% vs. 7.15%). For high frequency alleles, our results show similarity to neighbouring countries. However, 15 alleles (such as A*02:04, A*02:06, A*02:11 and A*02:17) found in our cohort in low frequencies were never reported in some or all neighbouring countries. This is the first report on HLA-A allele frequencies in Syria. In spite of the relatively low number of tested subjects, our results revealed a high degree of diversity, with 32 different alleles, reflecting the high ethnic heterogeneity of the Syrian population. The identification of alleles rarely or never reported in neighbouring countries indicates a higher genetic diversity in Syria.

  17. Mitochondrial DNA sequence-based phylogenetic relationship of Trichiurus lepturus (Perciformes: Trichiuridae) from the Persian Gulf

    PubMed Central

    Tamadoni Jahromi, S.; Mohd Noor, S. A.; Pirian, K.; Dehghani, R.; Nazemi, M.; Khazaali, A.

    2016-01-01

    In this study, mitochondrial DNA analysis using 16S ribosomal DNA (rDNA) was performed to investigate the phylogeny relationship of Trichiurus lepturus in the Persian Gulf compared to the other investigated area. The amplification of 16S rDNA resulted in a product of 600 bp in all samples. The results showed that the isolated strain belongs to T. lepturus showing 42 divergence sites among the same reported partial sequences of 16S rRNA gene from the other area (West Atlantic and Indo-Pacific area). Phylogeny results showed that all 18 haplotypes of the species clustered into five clades with reasonably high bootstrap support of values (>64%). Overall, the tree topology for both phylogenetic and phenetic trees for 16S rDNA was similar. Both trees exposed two major clusters, one wholly containing the haplotypes of the T. lepturus species belonging to Indo-Pacific area with two major sister groups including Persian Gulf specimen and the other cleared the Western Atlantic and Japan individuals clustered in another distinct clade supporting the differentiation between the two areas. Phylogenic relationship observed between the Persian Gulf and the other Indo-Pacific Individuals suggested homogeneity between two mentioned areas. PMID:27822250

  18. Post-Fragmentation Whole Genome Amplification-Based Method

    NASA Technical Reports Server (NTRS)

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

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have

  19. Attomolar quantitation of Mycobacterium tuberculosis by asymmetric helicase-dependent isothermal DNA-amplification and electrochemical detection.

    PubMed

    Barreda-García, Susana; González-Álvarez, María José; de-los-Santos-Álvarez, Noemí; Palacios-Gutiérrez, Juan José; Miranda-Ordieres, Arturo J; Lobo-Castañón, María Jesús

    2015-06-15

    A highly sensitive and robust method for the quantification of specific DNA sequences based on coupling asymmetric helicase-dependent DNA amplification to electrochemical detection is described. This method relies on the entrapment of the amplified ssDNA sequences on magnetic beads followed by a post-amplification hybridization assay to provide an added degree of specificity. As a proof-of-concept a 84-bases long sequence specific of Mycobacterium tuberculosis is amplified at 65°C, providing 3×10(6) amplification after 90 min. Using this system 0.5 aM, corresponding to 15 copies of the target gene in 50 µL of sample, can be successfully detected and reliably quantified under isothermal conditions in less than 4h. The assay has been applied to the detection of M. tuberculosis in sputum, pleural fluid and urine samples. Besides this application, the proposed assays is a powerful and general tool for molecular diagnostic that can be applied to the detection of other specific DNA sequences, taking full advantage of the plethora of genomic information now available.

  20. Amplification of postwildfire peak flow by debris

    USGS Publications Warehouse

    Kean, Jason W.; Mcguire, Luke; Rengers, Francis; Smith, Joel B.; Staley, Dennis M.

    2016-01-01

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

  1. Amplification of postwildfire peak flow by debris

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  2. Tyramide Signal Amplification for Immunofluorescent Enhancement.

    PubMed

    Faget, Lauren; Hnasko, Thomas S

    2015-01-01

    Enzyme-linked signal amplification is a key technique used to enhance the immunohistochemical detection of protein, mRNA, and other molecular species. Tyramide signal amplification (TSA) is based on a catalytic reporter deposit in close vicinity to the epitope of interest. The advantages of this technique are its simplicity, enhanced sensitivity, high specificity, and compatibility with modern multi-label fluorescent microscopy. Here, we describe the use of a TSA kit to increase the signal of enhanced green fluorescent protein (eGFP) expressed under the control of Slc17a6 regulatory elements in the brain of a transgenic mouse. The labeling procedure consists of 6 basic steps: (1) tissue preparation, (2) blocking of nonspecific epitopes, (3) binding with primary antibody, (4) binding with horseradish peroxidase-conjugated secondary antibody, (5) reacting with fluorescent tyramide substrate, and (6) imaging of the signal. The procedures described herein detail these steps and provide additional guidance and background to assist novice users.

  3. Weak-value amplification: state of play

    NASA Astrophysics Data System (ADS)

    Knee, George C.; Combes, Joshua; Ferrie, Christopher; Gauger, Erik M.

    2016-01-01

    Weak values arise in quantum theory when the result of a weak measurement is conditioned on a subsequent strong measurement. The majority of the trials are discarded, leaving only very few successful events. Intriguingly those can display a substantial signal amplification. This raises the question of whether weak values carry potential to improve the performance of quantum sensors, and indeed a number of impressive experimental results suggested this may be the case. By contrast, recent theoretical studies have found the opposite: using weak-values to obtain an amplification generally worsens metrological performance. This survey summarises the implications of those studies, which call for a reappraisal of weak values' utility and for further work to reconcile theory and experiment.

  4. Parametric nanomechanical amplification at very high frequency.

    PubMed

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

    2009-09-01

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

  5. Quantum amplification effect in a horizon fluctuation

    SciTech Connect

    Ansari, Mohammad H.

    2010-05-15

    The appearance of a few unevenly spaced bright flashes of light on top of Hawking radiation is the sign of the amplification effect in black hole horizon fluctuations. Previous studies on this problem suffer from the lack of considering all emitted photons in the theoretical spectroscopy of these fluctuations. In this paper, we include all of the physical transition weights and present a consistent intensity formula. This modifies a black hole radiation pattern.

  6. Hormonal Involvement in Breast Cancer Gene Amplification

    DTIC Science & Technology

    2008-10-01

    re-replication creates extra copies of the gene. This in turn will also increase production of the protein encoded by the amplified gene. Hormonal... increases in MCM proteins and Cdt1 have been shown to induce DNA amplification in yeast (Gopalakrishnan et al., 2001; Nguyen et al., 2001; Green et al...2006) and increased Cdt1 results in re-replication in human cells (Dorn et al., 2008). The N- terminus of Cdt1 is important for re-replication

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

    PubMed Central

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

    2017-01-01

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

  8. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems.

  9. Amplification of hofmeister effect by alcohols.

    PubMed

    Xu, Yun; Liu, Guangming

    2014-07-03

    We have demonstrated that Hofmeister effect can be amplified by adding alcohols to aqueous solutions. The lower critical solution temperature behavior of poly(N-isopropylacrylamide) has been employed as the model system to study the amplification of Hofmeister effect. The alcohols can more effectively amplify the Hofmeister effect following the series methanol < ethanol < 1-propanol < 2-propanol for the monohydric alcohols and following the series d-sorbitol ≈ xylitol ≈ meso-erythritol < glycerol < ethylene glycol < methanol for the polyhydric alcohols. Our study reveals that the relative extent of amplification of Hofmeister effect is determined by the stability of the water/alcohol complex, which is strongly dependent on the chemical structure of alcohols. The more stable solvent complex formed via stronger hydrogen bonds can more effectively differentiate the anions through the anion-solvent complex interactions, resulting in a stronger amplification of Hofmeister effect. This study provides an alternative method to tune the relative strength of Hofmeister effect besides salt concentration.

  10. Thermoelectric amplification of phonons in graphene

    NASA Astrophysics Data System (ADS)

    Dompreh, K. A.; Mensah, N. G.; Mensah, S. Y.; Fosuhene, S. K.

    2016-06-01

    Amplification of acoustic in-plane phonons due to an external temperature gradient (∇T) in single-layer graphene (SLG) was studied theoretically. The threshold temperature gradient (∇ T ) 0 g and the threshold voltage (V T ) 0 g in SLG were evaluated. For T = 77 K , the calculated value for (∇ T ) 0 g = 746.8 K / cm and (V T ) 0 g = 6.6 mV . The calculation was done in the hypersound regime. Further, the dependence of the normalized amplification ( Γ / Γ 0 ) on the frequency ω q and ∇ T / T were evaluated numerically and presented graphically. The calculated threshold temperature gradient (V T ) 0 g for SLG was higher than that obtained for homogeneous semiconductors (n-InSb) (∇ T ) 0 hom ≈ 10 3 K / cm , superlattices (∇ T ) 0 S L ≈ 384 K / cm , and cylindrical quantum wire (∇ T ) 0 c q w ≈ 10 2 K / cm . This makes SLG a much better material for thermoelectric phonon amplification.

  11. Estimating site amplification factors from ambient noise

    NASA Astrophysics Data System (ADS)

    Taylor, Steven R.; Gerstoft, Peter; Fehler, Michael C.

    2009-05-01

    We present a methodology to obtain frequency-dependent relative site amplification factors using ambient seismic noise. We treat a seismic network or array as a forced damped harmonic oscillator system where each station responds to a forcing function obtained from frequency-wavenumber beams of the ambient noise field. A network or array beam is necessary to estimate the forcing function. Taken over long time periods, each station responds to the forcing function showing a frequency-dependent resonance peak whose amplitude and spectral width depends upon the elastic and anelastic properties of the underlying medium. Our results are encouraging in that hard rock sites show little variability and have narrower resonance peaks with reduced amplitudes relative to soft rock sites in sedimentary basins. There is much more variability observed at soft rock sites and a tendency for spectral peaks to shift to higher frequencies and become broader as the site amplification increases. This could be due to due to lower densities and/or small-strain nonlinearity at stations having high site amplification.

  12. Optimized thermal amplification in a radiative transistor

    NASA Astrophysics Data System (ADS)

    Prod'homme, Hugo; Ordonez-Miranda, Jose; Ezzahri, Younes; Drevillon, Jeremie; Joulain, Karl

    2016-05-01

    The thermal performance of a far-field radiative transistor made up of a VO2 base in between a blackbody collector and a blackbody emitter is theoretically studied and optimized. This is done by using the grey approximation on the emissivity of VO2 and deriving analytical expressions for the involved heat fluxes and transistor amplification factor. It is shown that this amplification factor can be maximized by tuning the base temperature close to its critical one, which is determined by the temperature derivative of the VO2 emissivity and the equilibrium temperatures of the collector and emitter. This maximization is the result of the presence of two bi-stable temperatures appearing during the heating and cooling processes of the VO2 base and enables a thermal switching (temperature jump) characterized by a sizeable variation of the collector-to-base and base-to-emitter heat fluxes associated with a slight change of the applied power to the base. This switching effect leads to the optimization of the amplification factor and therefore it could be used for thermal modulation purposes.

  13. Comparison of HER2 gene amplification and KRAS alteration in eyelid sebaceous carcinomas with that in other eyelid tumors.

    PubMed

    Kwon, Mi Jung; Shin, Hyung Sik; Nam, Eun Sook; Cho, Seong Jin; Lee, Min Joung; Lee, Samuel; Park, Hye-Rim

    2015-05-01

    Eyelid sebaceous carcinoma (SC) represents a highly aggressive malignancy. Despite the poor prognosis, genetic alterations as potential molecular targets are not available. KRAS mutation and HER2 gene amplification may be candidates related to their genetic alterations. We examined the HER2 and KRAS alteration status in eyelid SCs and compared it with that in other eyelid tumors. The controversial topics of the human papillomavirus (HPV) and p16 expression were also investigated. HER2 amplification was determined by silver in situ hybridization, while immunohistochemistry was performed to study protein expressions in 14 SCs and controls, including 23 other eyelid malignancies and 14 benign tumors. Peptide nucleic acid-mediated PCR clamping and direct sequencing were used to detect KRAS mutations. HER2 protein overexpression was observed in 85.7% (12/14) of the SCs, of which two-thirds showed HER2 gene amplification. HER2 protein overexpression and HER2 amplification were found more frequently in eyelid SCs than in other eyelid tumors. All SCs harbored wild type KRAS genes. No HPV infections were identified in the SCs. Nevertheless, p16 overexpression was found in 71.4% (10/14) of SCs, irrespective of the status of HPV infection. Furthermore, p16 overexpression in eyelid SCs was also significantly higher than that in other eyelid tumors. HER2 protein overexpression, HER2 gene amplifications, and wild type KRAS genes are common in eyelid SCs. HER2 gene amplification may represent potential therapeutic targets for the treatment of eyelid SCs.

  14. Study Unveils New Method for Universal Extraction and PCR Amplification of Fungal DNA

    DTIC Science & Technology

    2014-06-12

    rare or hard to identify fungal infections. The new extraction and amplification method can be universally applied to fungi , according to the...best treatments. In addition, rare fungi , or species with phenotypic doppelgangers, can stump medical mycologists, so molecular methods are critical...However, isolating DNA from fungi can be problematic, and an inexpensive method to isolate and amplify nucleic acids from all types of pathogenic fungi

  15. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    PubMed Central

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-01-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20–100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. PMID:26050646

  16. A Sweet Spot for Molecular Diagnostics: Coupling Isothermal Amplification and Strand Exchange Circuits to Glucometers

    NASA Astrophysics Data System (ADS)

    Du, Yan; Hughes, Randall A.; Bhadra, Sanchita; Jiang, Yu Sherry; Ellington, Andrew D.; Li, Bingling

    2015-06-01

    Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.

  17. Enantioenrichment in sublimed amino acid mixtures.

    PubMed

    Viedma, Cristóbal; Ortiz, José E; de Torres, Trinidad; Cintas, Pedro

    2012-04-14

    A real amplification of an initial enantiomeric excess can be detected when two amino acids are sublimed at high temperature, even if one of the components is a racemic compound that does not convert into a conglomerate by sublimation.

  18. Ultrasensitive DNA detection based on two-step quantitative amplification on magnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Jin, Mingliang; Liu, Xia; van den Berg, Albert; Zhou, Guofu; Shui, Lingling

    2016-08-01

    Sensitive detection of a specific deoxyribo nucleic acid (DNA) sequence is important for biomedical applications. In this report, a two-step amplification strategy is developed based on magnetic nanoparticles (MNPs) to achieve ultrasensitive DNA fluorescence detection. The first level amplification is obtained from multiple binding sites on MNPs to achieve thousands of probe DNA molecules on one nanoparticle surface. The second level amplification is gained by enzymatic reaction to achieve fluorescence signal enhancement. MNPs functionalized by probe DNA (DNAp) are bound to target DNA (t-DNA) molecules with a ratio of 1:1 on a substrate with capture DNA (DNAc). After the MNPs with DNAp are released from the substrate, alkaline phosphatase (AP) is labelled to MNPs via hybridization reaction between DNAp on MNPs and detection DNAs (DNAd) with AP. The AP on MNPs catalyses non-fluorescent 4-methylumbelliferyl phosphate (4-MUP) to fluorescent 4-methylumbelliferone (4-MU) with high intensity. Finally, fluorescence intensity of the 4-MU is detected by a conventional fluorescence spectrophotometer. With this two-step amplification strategy, the limit of detection (LOD) of 2.8 × 10-18 mol l-1 for t-DNA has been achieved.

  19. All Three Rows of Outer Hair Cells Are Required for Cochlear Amplification.

    PubMed

    Murakoshi, Michio; Suzuki, Sho; Wada, Hiroshi

    2015-01-01

    In the mammalian auditory system, the three rows of outer hair cells (OHCs) located in the cochlea are thought to increase the displacement amplitude of the organ of Corti. This cochlear amplification is thought to contribute to the high sensitivity, wide dynamic range, and sharp frequency selectivity of the hearing system. Recent studies have shown that traumatic stimuli, such as noise exposure and ototoxic acid, cause functional loss of OHCs in one, two, or all three rows. However, the degree of decrease in cochlear amplification caused by such functional losses remains unclear. In the present study, a finite element model of a cross section of the gerbil cochlea was constructed. Then, to determine effects of the functional losses of OHCs on the cochlear amplification, changes in the displacement amplitude of the basilar membrane (BM) due to the functional losses of OHCs were calculated. Results showed that the displacement amplitude of the BM decreases significantly when a single row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification.

  20. Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform

    PubMed Central

    Long, Yuyin; Zhou, Cuisong; Wang, Congmin; Cai, Honglian; Yin, Cuiyun; Yang, Qiufang; Xiao, Dan

    2016-01-01

    Methodologies to detect disease biomarkers at ultralow concentrations can potentially improve the standard of living. A facile and label-free multi-amplification strategy is proposed for the ultrasensitive visual detection of HIV DNA biomarkers in real physiological media. This multi-amplification strategy not only exhibits a signficantly low detection limit down to 4.8 pM but also provides a label-free, cost-effective and facile technique for visualizing a few molecules of nucleic acid analyte with the naked eye. Importantly, the biosensor is capable of discriminating single-based mismatch lower than 5.0 nM in human serum samples. Moreover, the visual sensing platform exhibits excellent specificity, acceptable reusability and a long-term stability. All these advantages could be attributed to the nanofibrous sensing platform that 1) has a high surface-area-to-volume provided by electrospun nanofibrous membrane, and 2) combines glucose oxidase (GOx) biocatalysis, DNAzyme-catalyzed colorimetric reaction and catalytic hairpin assembly (CHA) recycling amplification together. This multi-amplification nanoplatform promises label-free and visual single-based mismatch DNA monitoring with high sensitivity and specificity, suggesting wide applications that range from virus detection to genetic disease diagnosis. PMID:27032385

  1. On the role of temperature feedbacks for Arctic amplification

    NASA Astrophysics Data System (ADS)

    Pithan, Felix; Mauritsen, Thorsten

    2013-04-01

    The amplification of global climate changes at the poles is a well-known feature of the climate system mentioned already by Arrhenius (1896). It has been linked to the surface-albedo feedback, changes in atmospheric and oceanic heat convergence, water vapour and cloud feedbacks and the albedo effect of black carbon on snow (Serreze and Barry, 2011). We here focus on the role of temperature feedbacks, which have received rather little attention in recent debates. The basic temperature feedback is the Planck feedback or the increase in the Earth's blackbody radiation due to a uniform temperature increase. Since the blackbody radiation scales with the fourth power of temperature, stronger warming is necessary in cold regions to balance a globally uniform radiative forcing. The second temperature feedback is caused by changes in the vertical atmospheric temperature structure: In the Tropics, deep convection leads to warming aloft being larger than at the surface, which causes a greater increase in outgoing longwave radiation compared a vertically uniform forcing and thus constitutes a negative feedback mechanism. In the Arctic, where warming is amplified at the surface, the lapse-rate feedback is positive (Wetherald and Manabe, 1975). We use CMIP5 model output and radiative Kernels to investigate the zonal distribution of temperature feedbacks. Arrhenius, S. (1896). On the influence of carbonic acid in the air upon the temperature of the ground Philos. Mag. J. Sci., 5, pp. 237-276 Serreze, M.C. and Barry, R.G. (2011) . Processes and impacts of Arctic amplification: A research synthesis, Global and Planetary Change, 77(1-2), pp. 85-96 Wetherald, R. and Manabe, S. (1975). The effects of changing the solar constant on the climate of a general circulation model. J. Atmos. Sci., 23 pp 2044-2059

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

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Faye, Oumar; Thaloengsok, Sasikanya; Heidenreich, Doris; Matangkasombut, Ponpan; Manopwisedjaroen, Khajohnpong; Sakuntabhai, Anavaj; Sall, Amadou A.; Hufert, Frank T.; Weidmann, Manfred

    2015-01-01

    Background Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. Methodology/Principal Findings Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38°C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. Conclusions/Significance During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations. PMID:26075598

  3. Nucleic acid arrays and methods of synthesis

    DOEpatents

    Sabanayagam, Chandran R.; Sano, Takeshi; Misasi, John; Hatch, Anson; Cantor, Charles

    2001-01-01

    The present invention generally relates to high density nucleic acid arrays and methods of synthesizing nucleic acid sequences on a solid surface. Specifically, the present invention contemplates the use of stabilized nucleic acid primer sequences immobilized on solid surfaces, and circular nucleic acid sequence templates combined with the use of isothermal rolling circle amplification to thereby increase nucleic acid sequence concentrations in a sample or on an array of nucleic acid sequences.

  4. Amplification of signaling events in bacteria.

    PubMed

    Dahlquist, Frederick W

    2002-05-14

    Bacteria respond to extremely shallow chemical gradients by modifying their motility in a process called chemotaxis. This chemotactic response is characterized by high sensitivity to small concentration differences, which extends over a large range of concentrations. This combination of high signal gain and large dynamic range results from both a memory of past events and the ability to amplify small differences in signal between the memory and the current environment. Dahlquist describes the signaling mechanism used by bacteria to regulate the flagellar motor and the places in this pathway where signal amplification may occur.

  5. SITE AMPLIFICATION OF EARTHQUAKE GROUND MOTION.

    USGS Publications Warehouse

    Hays, Walter W.

    1986-01-01

    When analyzing the patterns of damage in an earthquake, physical parameters of the total earthquake-site-structure system are correlated with the damage. Soil-structure interaction, the cause of damage in many earthquakes, involves the frequency-dependent response of both the soil-rock column and the structure. The response of the soil-rock column (called site amplification) is controversial because soil has strain-dependent properties that affect the way the soil column filters the input body and surface seismic waves, modifying the amplitude and phase spectra and the duration of the surface ground motion.

  6. Magnetic flux amplification by Lenz lenses

    NASA Astrophysics Data System (ADS)

    Schoenmaker, J.; Pirota, K. R.; Teixeira, J. C.

    2013-08-01

    Tailoring magnetic flux distribution is highly desirable in a wide range of applications such as magnetic sensors and biomedicine. In this paper we study the manipulation of induced currents in passive devices in order to engineer the distribution of magnetic flux intensity in a given region. We propose two different approaches, one based on especially designed wire loops (Lenz law) and the other based on solid conductive pieces (eddy currents). The gain of such devices is mainly determined by geometry giving perspective of high amplification. We consistently modeled, simulated, and executed the proposed devices. Doubled magnetic flux intensity is demonstrated experimentally for a moderate aspect ratio.

  7. Raman Amplification in Plasma: Thermal Effects

    SciTech Connect

    Farmer, John; Ersfeld, Bernhard; Jaroszynski, Dino

    2009-01-22

    The impact of thermal effects on Raman amplification in plasma is investigated theoretically. It is shown that damping and the shift in plasma resonance at finite temperature can alter the evolution of the amplified pulse and lead to pulse compression which is not predicted by the cold plasma model. Although thermal effects can lead to a reduction in the efficiency of the interaction, this can be ameliorated by using a chirped pump. In this case thermal effects can be beneficial and suppress the development of the train of pulses that develops behind the amplified pulse, as observed in the cold plasma model.

  8. Free electron laser designs for laser amplification

    DOEpatents

    Prosnitz, Donald; Szoke, Abraham

    1985-01-01

    Method for laser beam amplification by means of free electron laser techniques. With wiggler magnetic field strength B.sub.w and wavelength .lambda..sub.w =2.pi./k.sub.w regarded as variable parameters, the method(s) impose conditions such as substantial constancy of B.sub.w /k.sub.w or k.sub.w or B.sub.w and k.sub.w (alternating), coupled with a choice of either constant resonant phase angle or programmed phase space "bucket" area.

  9. Modeling Site Amplification in Eastern North America

    NASA Astrophysics Data System (ADS)

    Braganza, S.; Atkinson, G. M.; Ghofrani, H.; Hassani, B.

    2015-12-01

    A critical component in the understanding and interpretation of earthquake ground motions is the role that site effects play. In many parts of eastern North America, the soil layers which overlie glaciated bedrock produce strong and highly variable site responses. We use horizontal-to-vertical (H/V) response spectral ratios as the indicator variable by which to characterize the salient characteristics of site response in eastern Canada. We show that site response can be modeled using two descriptive variables that are readily obtainable: (i) peak resonant frequency (fpeak), as determined from H/V or depth-to-bedrock; and (ii) overall soil type (or stiffness). We use these variables to create a model of site amplification that can be used in the development of ground-motion prediction equations (GMPEs) and in real-time interactive ground-motion (IGM) map applications. The key to the site characterization is the relationship between fpeak and drift thickness (depth-to-bedrock), which we derive using H/V data from earthquakes in the region, combined with a detailed digital drift thickness map available online from the Ontario Geological Survey (OGS). The OGS map also provides information on soil type, which is correlated with peak amplitudes (Apeak) of response. We extend the study area to the city of Montreal using similar information from Chouinard and Rosset (2012). H/V spectral shapes may be associated with four main soil categories, which in decreasing order of stiffness are: bedrock, till, sand/clay, and organic soil/fill. The value of Apeak increases as stiffness decreases. We model site response by defining a generic site amplification curve, which is dependent only on fpeak and soil type. The generic curve enables an estimate of site amplification to be made over the entire frequency band of 0.1 to 50 Hz, knowing just the soil thickness and type. These site amplification curves can be applied in the development of regional GMPEs, and in the construction of

  10. Internal entanglement amplification by external interactions

    SciTech Connect

    Peskin, Uri; Huang Zhen; Kais, Sabre

    2007-07-15

    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 demonstrated. The possibility of entangling noninteracting spins in a stationary state is also demonstrated by coupling each one of them to a flying qubit in a quantum wire.

  11. Amplification and characterization of eukaryotic structural genes.

    PubMed

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

    1978-05-01

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

  12. Bilateral amplification and sound localization: then and now.

    PubMed

    Simon, Helen J

    2005-01-01

    This article is concerned with the evolution and pros and cons of bilateral amplification. Determining whether a bilateral hearing aid fitting is superior to that of a monaural hearing aid is a long-standing question; for this reason, the trend toward bilateral amplification has been slow. However, it is now assumed that bilateral amplification has significant advantages over monaural amplification in most cases, a view that is supported by our localization results. In this article, we will address the advantages of bilateral hearing aids and reveal some new localization data that show that most listeners with bilateral amplification, when tested unaided, as well as normal-hearing listeners manifested very high degrees of symmetry in their judgments of perceived angle while listeners who routinely use monaural amplification and those with asymmetric hearing loss had relatively large asymmetries. These data show that asymmetry in localization judgments is a much more sensitive indicator of abnormal localization ability than the magnitude of localization errors.

  13. Specific replication origins promote DNA amplification in fission yeast.

    PubMed

    Kiang, Lee; Heichinger, Christian; Watt, Stephen; Bähler, Jürg; Nurse, Paul

    2010-09-15

    To ensure equal replication of the genome in every eukaryotic cell cycle, replication origins fire only once each S phase and do not fire after passive replication. Failure in these controls can lead to local amplification, contributing to genome instability and the development of cancer. To identify features of replication origins important for such amplification, we have investigated origin firing and local genome amplification in the presence of excess helicase loaders Cdc18 and Cdt1 in fission yeast. We find that S phase controls are attenuated and coordination of origin firing is lost, resulting in local amplification. Specific origins are necessary for amplification but act only within a permissive chromosomal context. Origins associated with amplification are highly AT-rich, fire efficiently and early during mitotic S phase, and are located in large intergenic regions. We propose that these features predispose replication origins to re-fire within a single S phase, or to remain active after passive replication.

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

    SciTech Connect

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

    2009-11-01

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

  15. In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

    PubMed Central

    O‘Connor, Matthew J.; Bishop, Keith; Workman, Robert G.; Maddison, Ben C.

    2017-01-01

    ABSTRACT The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrPSc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrPSc from 3 distinct experimental isolates of H-BSE, could detect PrPSc in as little as 1×10−12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrPSc with distinct protease K-resistance and protease K-resistant PrPSc molecular weights. PMID:28281929

  16. Explanatory model for sound amplification in a stethoscope

    NASA Astrophysics Data System (ADS)

    Eshach, H.; Volfson, A.

    2015-01-01

    In the present paper we suggest an original physical explanatory model that explains the mechanism of the sound amplification process in a stethoscope. We discuss the amplification of a single pulse, a continuous wave of certain frequency, and finally we address the resonant frequencies. It is our belief that this model may provide students with opportunities to not only better understand the amplification mechanism of a stethoscope, but also to strengthen their understanding of sound, pressure, waves, resonance modes, etc.

  17. Sequence-based discrimination of protein-RNA interacting residues using a probabilistic approach.

    PubMed

    Pai, Priyadarshini P; Dash, Tirtharaj; Mondal, Sukanta

    2017-04-07

    Protein interactions with ribonucleic acids (RNA) are well-known to be crucial for a wide range of cellular processes such as transcriptional regulation, protein synthesis or translation, and post-translational modifications. Identification of the RNA-interacting residues can provide insights into these processes and aid in relevant biotechnological manipulations. Owing to their eventual potential in combating diseases and industrial production, several computational attempts have been made over years using sequence- and structure-based information. Recent comparative studies suggest that despite these developments, many problems are faced with respect to the usability, prerequisites, and accessibility of various tools, thereby calling for an alternative approach and perspective supplementation in the prediction scenario. With this motivation, in this paper, we propose the use of a simple-yet-efficient conditional probabilistic approach based on the application of local occurrence of amino acids in the interacting region in a non-numeric sequence feature space, for discriminating between RNA interacting and non-interacting residues. The proposed method has been meticulously tested for robustness using a cross-estimation method showing MCC of 0.341 and F- measure of 66.84%. Upon exploring large scale applications using benchmark datasets available to date, this approach showed an encouraging performance comparable with the state-of-art. The software is available at https://github.com/ABCgrp/DORAEMON.

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

    PubMed

    Zhao, Lue Ping; Huang, Xin

    2013-07-01

    The advent of next-generation sequencing technologies affords the ability to sequence thousands of subjects cost-effectively, and is revolutionizing the landscape of genetic research. With the evolving genotyping/sequencing technologies, it is not unrealistic to expect that we will soon obtain a pair of diploidic fully phased genome sequences from each subject in the near future. Here, in light of this potential, we propose an analytic framework called, recursive organizer (ROR), which recursively groups sequence variants based upon sequence similarities and their empirical disease associations, into fewer and potentially more interpretable super sequence variants (SSV). As an illustration, we applied ROR to assess an association between HLA-DRB1 and type 1 diabetes (T1D), discovering SSVs of HLA-DRB1 with sequence data from the Wellcome Trust Case Control Consortium. Specifically, ROR reduces 36 observed unique HLA-DRB1 sequences into 8 SSVs that empirically associate with T1D, a fourfold reduction of sequence complexity. Using HLA-DRB1 data from Type 1 Diabetes Genetics Consortium as cases and data from Fred Hutchinson Cancer Research Center as controls, we are able to validate associations of these SSVs with T1D. Further, SSVs consist of nine nucleotides, and each associates with its corresponding amino acids. Detailed examination of these selected amino acids reveals their potential functional roles in protein structures and possible implication to the mechanism of T1D.

  19. A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features.

    PubMed

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

    2017-02-01

    Palmitoylation is the covalent attachment of lipids to amino acid residues in proteins. As an important form of protein posttranslational modification, it increases the hydrophobicity of proteins, which contributes to the protein transportation, organelle localization, and functions, therefore plays an important role in a variety of cell biological processes. Identification of palmitoylation sites is necessary for understanding protein-protein interaction, protein stability, and activity. Since conventional experimental techniques to determine palmitoylation sites in proteins are both labor intensive and costly, a fast and accurate computational approach to predict palmitoylation sites from protein sequences is in urgent need. In this study, a support vector machine (SVM)-based method was proposed through integrating PSI-BLAST profile, physicochemical properties, [Formula: see text]-mer amino acid compositions (AACs), and [Formula: see text]-mer pseudo AACs into the principal feature vector. A recursive feature selection scheme was subsequently implemented to single out the most discriminative features. Finally, an SVM method was implemented to predict palmitoylation sites in proteins based on the optimal features. The proposed method achieved an accuracy of 99.41% and Matthews Correlation Coefficient of 0.9773 for a benchmark dataset. The result indicates the efficiency and accuracy of our method in prediction of palmitoylation sites based on protein sequences.

  20. Comparison of sensor structures for the signal amplification of surface plasmon resonance immunoassay using enzyme precipitation

    NASA Astrophysics Data System (ADS)

    Yang, Chih-Tsung; Thierry, Benjamin

    2015-12-01

    Surface plasmon resonance (SPR) biosensing has been successfully applied for the label-free detection of a broad range of bioanalytes ranging from bacteria, cell, exosome, protein and nucleic acids. When it comes to the detection of small molecules or analytes found at low concentration, amplification schemes are desirable to enhance binding signals and in turn increase sensitivity. A number of SPR signal amplification schemes have been developed and validated; however, little effort has been devoted to understanding the effect of the SPR sensor structures on the amplification of binding signals and therefore on the overall sensing performance. The physical phenomenon of long-range SPR (LRSPR) relies on the propagation of coupled surface plasmonic waves on the opposite sides of a nanoscale-thick noble metal film suspended between two dielectrics with similar refractive indices. Importantly, as compared with commonly used conventional SPR (cSPR), LRSPR is not only characterized by a longer penetration depth of the plasmonic waves in the analyzed medium but also by a greater sensitivity to bulk refractive index changes. In this work, an immunoassay signal amplification platform based on horseradish peroxidase (HRP) catalyzed precipitation was utilized to investigate the sensing performance with regards to cSPR and LRSPR. The enzymatic precipitation of 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 was used to amplify SPR signals. The structure-function relationship of cSPR and LRSPR sensors is presented for both standard refractometric measurements and the enzymatic precipitation scheme. Experimental data shows that despite its inherent higher sensitivity to bulk refractive index changes and higher figure of merit, LRSPR was characterized by a lower angular sensitivity in the model enzymatic amplification scheme used here.

  1. Space Optical Communications Using Laser Beam Amplification

    NASA Technical Reports Server (NTRS)

    Agrawal, Govind

    2015-01-01

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

  2. ras gene Amplification and malignant transformation.

    PubMed Central

    Pulciani, S; Santos, E; Long, L K; Sorrentino, V; Barbacid, M

    1985-01-01

    Morphologic transformation of NIH 3T3 mouse cells occurs upon transfection of these cells with large amounts (greater than or equal to 10 micrograms) of recombinant DNA molecules carrying the normal human H-ras-1 proto-oncogene. We provide experimental evidence indicating that transformation of these NIH 3T3 cells results from the combined effect of multiple copies of the H-ras-1 proto-oncogene rather than from spontaneous mutation of one of the transfected H-ras-1 clones (E. Santos, E.P. Reddy, S. Pulciani, R.J. Feldman, and M. Barbacid, Proc. Natl. Acad. Sci. USA 80:4679-4683, 1983). Levels of H-ras-1 RNA and p21 expression are highly elevated in the NIH 3T3 transformants, and in those cases examined, these levels correlate with the malignant properties of these cells. We have also investigated the presence of amplified ras genes in a variety of human carcinomas. In 75 tumor biopsies, we found amplification of the human K-ras-2 locus in one carcinoma of the lung. These results indicate that ras gene amplification is an alternative pathway by which ras genes may participate in the development of human neoplasia. Images PMID:3915535

  3. Induction of gene amplification in Plasmodium falciparum

    SciTech Connect

    Rogers, P.L.

    1985-01-01

    Human erythrocytic in vitro cultures of Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with /sup 35/S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.

  4. A PARAMETER STUDY FOR BAROCLINIC VORTEX AMPLIFICATION

    SciTech Connect

    Raettig, Natalie; Klahr, Hubert; Lyra, Wladimir E-mail: klahr@mpia.de

    2013-03-10

    Recent studies have shown that baroclinic vortex amplification is strongly dependent on certain factors, namely, the global entropy gradient, the efficiency of thermal diffusion and/or relaxation as well as numerical resolution. We conduct a comprehensive study of a broad range and combination of various entropy gradients, thermal diffusion and thermal relaxation timescales via local shearing sheet simulations covering the parameter space relevant for protoplanetary disks. We measure the Reynolds stresses as a function of our control parameters and see that there is angular momentum transport even for entropy gradients as low as {beta} = -dln s/dln r = 1/2. Values we expect in protoplanetary disks are between {beta} = 0.5-2.0 The amplification-rate of the perturbations, {Gamma}, appears to be proportional to {beta}{sup 2} and thus proportional to the square of the Brunt-Vaeisaelae frequency ({Gamma}{proportional_to}{beta}{sup 2}{proportional_to}N {sup 2}). The saturation level of Reynolds stresses, on the other hand, seems to be proportional to {beta}{sup 1/2}. This highlights the importance of baroclinic effects even for the low entropy gradients expected in protoplanetary disks.

  5. Experimental noiseless linear amplification using weak measurements

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  6. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    PubMed

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.

  7. Direct amplification of casework bloodstains using the Promega PowerPlex(®) 21 PCR amplification system.

    PubMed

    Gray, Kerryn; Crowle, Damian; Scott, Pam

    2014-09-01

    A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex(®) 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex(®) 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13μL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of

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

    PubMed

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

  9. Exonuclease III-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA.

    PubMed

    Zhou, Fulin; Li, Baoxin

    2015-07-21

    Detection of ultralow concentration of specific DNA sequence is a central challenge in the early diagnosis of gene-related disease and biodefense application. Herein, we report a dual-amplification strategy for highly sensitive fluorescence detection of DNA. In this proposed strategy, a dumbbell-shaped DNA probe is designed to integrate target binding, magnetic separation, and signal response. In the presence of specific DNA target, the multifunctional dumbbell probe can initiate exonuclease III (Exo III)-aided target recycling amplification, and, in the meantime, generate a large number of fluorescein (FAM)-encapsulated liposomes. The developed method offers very high sensitivity due to primary amplification via numerous FAM from a liposome and secondary amplification via target recycling amplification. The detection limit of the proposed method can reach 4 aM, which is much lower than that of the Exo III-aided target recycling technique applied for DNA quantification without FAM-encapsulated liposomes amplification. Moreover, the dual-signal amplification process can be completed one-step in this system. Therefore, this method provides a simple, isothermal, and low-cost approach for sensitive detection of DNA and holds a great potential for early diagnosis in gene-related diseases.

  10. Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate.

    PubMed

    Song, Weiling; Zhang, Qiao; Xie, Xuxu; Zhang, Shusheng

    2014-11-15

    In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6 × 10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

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

    NASA Astrophysics Data System (ADS)

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-09-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

  12. Amplification of chirality through self-replication of micellar aggregates in water.

    PubMed

    Bukhryakov, Konstantin V; Almahdali, Sarah; Rodionov, Valentin O

    2015-03-17

    We describe a system in which the self-replication of micellar aggregates results in a spontaneous amplification of chirality in the reaction products. In this system, amphiphiles are synthesized from two "clickable" fragments: a water-soluble "head" and a hydrophobic "tail". Under biphasic conditions, the reaction is autocatalytic, as aggregates facilitate the transfer of hydrophobic molecules to the aqueous phase. When chiral, partially enantioenriched surfactant heads are used, a strong nonlinear induction of chirality in the reaction products is observed. Preseeding the reaction mixture with an amphiphile of one chirality results in the amplification of this product and therefore information transfer between generations of self-replicating aggregates. Because our amphiphiles are capable of catalysis, information transfer, and self-assembly into bounded structures, they present a plausible model for prenucleic acid "lipid world" entities.

  13. Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics.

    PubMed

    James, Ameh; Macdonald, Joanne

    2015-01-01

    Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

  14. Signal Amplification of Bioassay Using Zinc Nanomaterials

    NASA Astrophysics Data System (ADS)

    Cowles, Chad L.

    An emerging trend in the analytical detection sciences is the employment of nanomaterials for bioassay signal transduction to identify analytes critical to public health. These nanomaterials have been specifically investigated for applications which require identification of trace levels of cells, proteins, or other molecules that can have broad ranging impacts to human health in fields such as clinical diagnostics, environmental monitoring, food and drink control, and the prevention of bioterrorism. Oftentimes these nanoparticle-based signal transduction or amplification approaches offer distinct advantages over conventional methods such as increased sensitivity, rapidity, or stability. The biological application of nanoparticles however, does suffer from drawbacks that have limited more widespread adoption of these techniques. Some of these drawbacks are, high cost and toxicity, arduous synthesis methods, functionalization and bioconjugation challenges, and laboratory disposal and environmental hazard issues, all of which have impeded the progression of this technology in some way or another. This work aims at developing novel techniques that offer solutions to a number of these hurdles through the development of new nanoparticle-based signal transduction approaches and the description of a previously undescribed nanomaterial. Zinc-based nanomaterials offer the opportunity to overcome some of the limitations that are encountered when other nanomaterials are employed for bioassay signal transduction. On the other hand, the biological application of zinc nanomaterials has been difficult because in general their fluorescence is in the blue range and the reported quantum yields are usually too low for highly sensitive applications. The advantages of using zinc nanomaterials for biological applications, such as reduced toxicity, simple synthesis, low cost, and straightforward functionalization strategies contribute to the research interest in their application as

  15. Coordinated movement of the three rows of outer hair cells is essential for cochlear amplification

    NASA Astrophysics Data System (ADS)

    Murakoshi, Michio; Suzuki, Sho; Wada, Hiroshi

    2015-12-01

    The process known as cochlear amplification is realized by coordinated movement of the outer hair cells (OHCs) in response to changes in their membrane potential. In this process, the displacement amplitude of the basilar membrane (BM) is thought to be increased, thereby leading to the high sensitivity, wide dynamic range and sharp frequency selectivity of our hearing. Unfortunately, however, OHCs are vulnerable to noise exposure, ototoxic acid, aging and so on. Previous studies have shown that exposure to intense noise causes functional loss of OHCs from the innermost row (i.e., close to the modiolus) to the outermost row (i.e., close to the cochlear wall). On the contrary, by other traumatic stimuli such as ototoxic acid, aging and ischemia, such loss of OHCs has been reported to occur from the outermost row toward the innermost row. However, how the cochlear amplification changes when coordinated movement of OHCs is impaired, that is when the OHCs in one, two or all three rows have become dysfunctional, remains unclear. In the present study, therefore, a finite element (FE) model of the gerbil cochlea, which takes the motility of OHCs into account, was developed based on our previous FE model. Using this model, changes in the displacement amplitude of the BM due to the functional loss of OHCs in one, two or all three rows were investigated and the effects of incoordination of the three rows of OHCs on cochlear amplification were estimated. Results showed that the displacement amplitude of the BM significantly decreased when either the innermost row or the outermost row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification.

  16. Sequence-Based Predictions of Lipooligosaccharide Diversity in the Neisseriaceae and Their Implication in Pathogenicity

    PubMed Central

    Stein, Daniel C.; Miller, Clinton J.; Bhoopalan, Senthil V.; Sommer, Daniel D.

    2011-01-01

    Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation. PMID:21533118

  17. Parametric amplification of a superconducting plasma wave

    NASA Astrophysics Data System (ADS)

    Rajasekaran, S.; Casandruc, E.; Laplace, Y.; Nicoletti, D.; Gu, G. D.; Clark, S. R.; Jaksch, D.; Cavalleri, A.

    2016-11-01

    Many applications in photonics require all-optical manipulation of plasma waves, which can concentrate electromagnetic energy on sub-wavelength length scales. This is difficult in metallic plasmas because of their small optical nonlinearities. Some layered superconductors support Josephson plasma waves, involving oscillatory tunnelling of the superfluid between capacitively coupled planes. Josephson plasma waves are also highly nonlinear, and exhibit striking phenomena such as cooperative emission of coherent terahertz radiation, superconductor-metal oscillations and soliton formation. Here, we show that terahertz Josephson plasma waves can be parametrically amplified through the cubic tunnelling nonlinearity in a cuprate superconductor. Parametric amplification is sensitive to the relative phase between pump and seed waves, and may be optimized to achieve squeezing of the order-parameter phase fluctuations or terahertz single-photon devices.

  18. Control and amplification of cortical neurodynamics

    NASA Astrophysics Data System (ADS)

    Liljenstroem, Hans; Aronsson, P.

    1999-03-01

    We investigate different mechanisms for the control and amplification of cortical neurodynamics, using a neural network model of a three layered cortical structure. We show that different dynamical states can be obtained by changing a control parameter of the input-output relation, or by changing the noise level. Point attractor, limit cycle, and strange attractor dynamics occur at different values of the control parameter. For certain, optimal noise levels, system performance is maximized, analogous to stochastic resonance phenomena. Noise can also be used to induce different dynamical states. A few noisy network units distributed in a network layer can result in global synchronous oscillations, or waves of activity moving across the network. We further demonstrate that fast synchronization of network activity can be obtained by implementing electromagnetic interactions between network units.

  19. Parametric amplification of a superconducting plasma wave

    SciTech Connect

    Rajasekaran, S.; Casandruc, E.; Laplace, Y.; Nicoletti, D.; Gu, G. D.; Clark, S. R.; Jaksch, D.; Cavalleri, A.

    2016-07-11

    Many applications in photonics require all-optical manipulation of plasma waves, which can concentrate electromagnetic energy on sub-wavelength length scales. This is difficult in metallic plasmas because of their small optical nonlinearities. Some layered superconductors support Josephson plasma waves, involving oscillatory tunnelling of the superfluid between capacitively coupled planes. Josephson plasma waves are also highly nonlinear, and exhibit striking phenomena such as cooperative emission of coherent terahertz radiation, superconductor–metal oscillations and soliton formation. In this paper, we show that terahertz Josephson plasma waves can be parametrically amplified through the cubic tunnelling nonlinearity in a cuprate superconductor. Finally, parametric amplification is sensitive to the relative phase between pump and seed waves, and may be optimized to achieve squeezing of the order-parameter phase fluctuations or terahertz single-photon devices.

  20. Parametric amplification by coupled flux qubits

    SciTech Connect

    Rehák, M.; Neilinger, P.; Grajcar, M.; Oelsner, G.; Hübner, U.; Meyer, H.-G.; Il'ichev, E.

    2014-04-21

    We report parametric amplification of a microwave signal in a Kerr medium formed from superconducting qubits. Two mutually coupled flux qubits, embedded in the current antinode of a superconducting coplanar waveguide resonator, are used as a nonlinear element. Shared Josephson junctions provide the qubit-resonator coupling, resulting in a device with a tunable Kerr constant (up to 3 × 10{sup −3}) and a measured gain of about 20 dB. This arrangement represents a unit cell which can be straightforwardly extended to a quasi one-dimensional quantum metamaterial with large tunable Kerr nonlinearity, providing a basis for implementation of wide-band travelling wave parametric amplifiers.

  1. Optimizing biased semiconductor superlattices for terahertz amplification

    SciTech Connect

    Lei, Xiaoli; Wang, Dawei; Wu, Zhaoxin; Dignam, M. M.

    2014-08-11

    Over the past 15 yr or more, researchers have been trying to achieve gain for electromagnetic fields in the terahertz frequency region using biased semiconductor superlattices, but with little success. In this work, we employ our model of the excitonic states in biased GaAs/Al{sub 0.3}Ga{sub 0.7}As semiconductor superlattices to find the optimal structures for amplification of terahertz radiation. In particular, we determine the optimum well width, barrier width, and bias field for terahertz fields with frequencies ranging from 1 to 4 terahertz. We find that gain coefficients on the order of 40 cm{sup −1} should be achievable over most of this frequency range.

  2. Amplification sans bruit d'images optiques

    NASA Astrophysics Data System (ADS)

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

    2004-11-01

    Nous utilisons un Oscillateur Paramétrique Optique (OPO) pompé sous le seuil dans le but d'amplifier une image multimode transverse sans dégradation du rapport signal à bruit. Le dispositif expérimental met en œuvre un OPO de type II triplement résonant et semi-confocal pour le faisceau amplifié. L'existence d'effets quantiques lors de l'amplification multimode dans un tel dispositif a été montrée expérimentalement. Plus généralement, ceci nous a amené à étudier les propriétés quantiques transverses des faisceaux lumineux amplifiés. Une telle étude peut trouver des applications non seulement en imagerie, mais également dans le traitement quantique de l'information.

  3. Dispersion compensation in chirped pulse amplification systems

    DOEpatents

    Bayramian, Andrew James; Molander, William A.

    2014-07-15

    A chirped pulse amplification system includes a laser source providing an input laser pulse along an optical path. The input laser pulse is characterized by a first temporal duration. The system also includes a multi-pass pulse stretcher disposed along the optical path. The multi-pass pulse stretcher includes a first set of mirrors operable to receive input light in a first plane and output light in a second plane parallel to the first plane and a first diffraction grating. The pulse stretcher also includes a second set of mirrors operable to receive light diffracted from the first diffraction grating and a second diffraction grating. The pulse stretcher further includes a reflective element operable to reflect light diffracted from the second diffraction grating. The system further includes an amplifier, a pulse compressor, and a passive dispersion compensator disposed along the optical path.

  4. Chirped pulse amplification: Present and future

    SciTech Connect

    Maine, P.; Strickland, D.; Pessot, M.; Squier, J.; Bado, P.; Mourou, G.; Harter, D.

    1988-01-01

    Short pulses with ultrahigh peak powers have been generated in Nd: glass and Alexandrite using the Chirped Pulse Amplification (CPA) technique. This technique has been successful in producing picosecond terawatt pulses with a table-top laser system. In the near future, CPA will be applied to large laser systems such as NOVA to produce petawatt pulses (1 kJ in a 1 ps pulse) with focused intensities exceeding 10/sup /plus/21/ W/cm/sup 2/. These pulses will be associated with electric fields in excess of 100 e/a/sub o//sup 2/ and blackbody energy densities equivalent to 3 /times/ 10/sup 10/ J/cm/sup 3/. This petawatt source will have important applications in x-ray laser research and will lead to fundamentally new experiments in atomic, nuclear, solid-state, plasma, and high-energy density physics. A review of present and future designs are discussed. 17 refs., 5 figs.

  5. Mechanisms of Gene Duplication and Amplification

    PubMed Central

    Reams, Andrew B.; Roth, John R.

    2015-01-01

    Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number. PMID:25646380

  6. Parametric amplification of a superconducting plasma wave

    DOE PAGES

    Rajasekaran, S.; Casandruc, E.; Laplace, Y.; ...

    2016-07-11

    Many applications in photonics require all-optical manipulation of plasma waves, which can concentrate electromagnetic energy on sub-wavelength length scales. This is difficult in metallic plasmas because of their small optical nonlinearities. Some layered superconductors support Josephson plasma waves, involving oscillatory tunnelling of the superfluid between capacitively coupled planes. Josephson plasma waves are also highly nonlinear, and exhibit striking phenomena such as cooperative emission of coherent terahertz radiation, superconductor–metal oscillations and soliton formation. In this paper, we show that terahertz Josephson plasma waves can be parametrically amplified through the cubic tunnelling nonlinearity in a cuprate superconductor. Finally, parametric amplification is sensitivemore » to the relative phase between pump and seed waves, and may be optimized to achieve squeezing of the order-parameter phase fluctuations or terahertz single-photon devices.« less

  7. Amplification of seismic waves by the Seattle basin, Washington State

    USGS Publications Warehouse

    Pratt, T.L.; Brocher, T.M.; Weaver, C.S.; Creager, K.C.; Snelson, C.M.; Crosson, R.S.; Miller, K.C.; Trehu, A.M.

    2003-01-01

    Recordings of the 1999 Mw 7.6 Chi-Chi (Taiwan) earthquake, two local earthquakes, and five blasts show seismic-wave amplification over a large sedimentary basin in the U.S. Pacific Northwest. For weak ground motions from the Chi-Chi earthquake, the Seattle basin amplified 0.2- to 0.8-Hz waves by factors of 8 to 16 relative to bedrock sites west of the basin. The amplification and peak frequency change during the Chi-Chi coda: the initial S-wave arrivals (0-30 sec) had maximum amplifications of 12 at 0.5-0.8 Hz, whereas later arrivals (35-65 sec) reached amplifications of 16 at 0.3-0.5 Hz. Analysis of local events in the 1.0- to 10.0-Hz frequency range show fourfold amplifications for 1.0-Hz weak ground motion over the Seattle basin. Amplifications decrease as frequencies increase above 1.0 Hz, with frequencies above 7 Hz showing lower amplitudes over the basin than at bedrock sites. Modeling shows that resonance in low-impedance deposits forming the upper 550 m of the basin beneath our profile could cause most of the observed amplification, and the larger amplification at later arrival times suggests surface waves also play a substantial role. These results emphasize the importance of shallow deposits in determining ground motions over large basins.

  8. Evaluating Sound Field Amplification Technology in New Brunswick Schools

    ERIC Educational Resources Information Center

    Rubin, Rhonda; Aquino-Russell, Catherine; Flagg-Williams, Joan

    2007-01-01

    (Purpose) The purpose of this study was to investigate the effects of classroom sound field amplification on communication in kindergarten through grade 3 classrooms. (Methodology) Sixty classrooms were involved in the study; half of the classrooms were provided with sound field amplification. The flow of communication was measured through…

  9. Unilateral versus bilateral amplification for adults with impaired hearing.

    PubMed

    Walden, Therese C; Walden, Brian E

    2005-09-01

    This study compared unilateral and bilateral aided speech recognition in background noise in 28 patients being fitted with amplification. Aided QuickSIN (Quick Speech-in-Noise test) scores were obtained for bilateral amplification and for unilateral amplification in each ear. In addition, right-ear directed and left-ear directed recall on the Dichotic Digits Test (DDT) was obtained from each participant. Results revealed that the vast majority of patients obtained better speech recognition in background noise on the QuickSIN from unilateral amplification than from bilateral amplification. There was a greater tendency for bilateral amplification to have a deleterious effect among older patients. Most frequently, better aided QuickSIN performance was obtained in the right ear of participants, despite similar hearing thresholds in both ears. Finally, patients tended to perform better on the DDT in the ear that provided less SNR loss on the QuickSIN. Results suggest that bilateral amplification may not always be beneficial in every daily listening environment when background noise is present, and it may be advisable for patients wearing bilateral amplification to remove one hearing aid when difficulty is encountered understanding speech in background noise.

  10. Explanatory Model for Sound Amplification in a Stethoscope

    ERIC Educational Resources Information Center

    Eshach, H.; Volfson, A.

    2015-01-01

    In the present paper we suggest an original physical explanatory model that explains the mechanism of the sound amplification process in a stethoscope. We discuss the amplification of a single pulse, a continuous wave of certain frequency, and finally we address the resonant frequencies. It is our belief that this model may provide students with…

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

    PubMed

    Littlejohn, Mathew D; Tiplady, Kathryn; Fink, Tania A; Lehnert, Klaus; Lopdell, Thomas; Johnson, Thomas; Couldrey, Christine; Keehan, Mike; Sherlock, Richard G; Harland, Chad; Scott, Andrew; Snell, Russell G; Davis, Stephen R; Spelman, Richard J

    2016-05-05

    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.

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

    PubMed Central

    Littlejohn, Mathew D.; Tiplady, Kathryn; Fink, Tania A.; Lehnert, Klaus; Lopdell, Thomas; Johnson, Thomas; Couldrey, Christine; Keehan, Mike; Sherlock, Richard G.; Harland, Chad; Scott, Andrew; Snell, Russell G.; Davis, Stephen R.; Spelman, Richard J.

    2016-01-01

    The mammary gland is a prolific lipogenic organ, synthesising copious amounts of triglycerides for secretion into milk. The fat content of milk varies widely both between and within species, and recent independent genome-wide association studies have highlighted a milk fat percentage quantitative trait locus (QTL) of large effect on bovine chromosome 5. Although both EPS8 and MGST1 have been proposed to underlie these signals, the causative status of these genes has not been functionally confirmed. To investigate this QTL in detail, we report genome sequence-based imputation and association mapping in a population of 64,244 taurine cattle. This analysis reveals a cluster of 17 non-coding variants spanning MGST1 that are highly associated with milk fat percentage, and a range of other milk composition traits. Further, we exploit a high-depth mammary RNA sequence dataset to conduct expression QTL (eQTL) mapping in 375 lactating cows, revealing a strong MGST1 eQTL underpinning these effects. These data demonstrate the utility of DNA and RNA sequence-based association mapping, and implicate MGST1, a gene with no obvious mechanistic relationship to milk composition regulation, as causally involved in these processes. PMID:27146958

  13. Mutualism Breakdown by Amplification of Wolbachia Genes

    PubMed Central

    Chrostek, Ewa; Teixeira, Luis

    2015-01-01

    Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on

  14. Mutualism breakdown by amplification of Wolbachia genes.

    PubMed

    Chrostek, Ewa; Teixeira, Luis

    2015-02-01

    Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on

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

    PubMed

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

    2014-04-01

    Kombucha is a sweetened tea beverage that, as a consequence of fermentation, contains ethanol, carbon dioxide, a high concentration of acid (gluconic, acetic and lactic) as well as a number of other metabolites and is thought to contain a number of health-promoting components. The sucrose-tea solution is fermented by a symbiosis of bacteria and yeast embedded within a cellulosic pellicle, which forms a floating mat in the tea, and generates a new layer with each successful fermentation. The specific identity of the microbial populations present has been the focus of attention but, to date, the majority of studies have relied on culture-based analyses. To gain a more comprehensive insight into the kombucha microbiota we have carried out the first culture-independent, high-throughput sequencing analysis of the bacterial and fungal populations of 5 distinct pellicles as well as the resultant fermented kombucha at two time points. Following the analysis it was established that the major bacterial genus present was Gluconacetobacter, present at >85% in most samples, with only trace populations of Acetobacter detected (<2%). A prominent Lactobacillus population was also identified (up to 30%), with a number of sub-dominant genera, not previously associated with kombucha, also being revealed. The yeast populations were found to be dominated by Zygosaccharomyces at >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.

  16. Topographical and geological amplification: case studies and engineering implications

    USGS Publications Warehouse

    Celebi, M.

    1991-01-01

    Topographical and geological amplification that occurred during past earthquakes are quantified using spectral ratios of recorded motions. Several cases are presented from the 1985 Chilean and Mexican earthquakes as well as the 1983 Coalinga (California) and 1987 Supersition Hills (California) earthquake. The strong motions recorded in Mexico City during the 1985 Michoacan earthquake are supplemented by ambient motions recorded within Mexico City to quantify the now well known resonating frequencies of the Mexico City lakebed. Topographical amplification in Canal Beagle (Chile), Coalinga and Superstition Hills (California) are quantified using the ratios derived from the aftershocks following the earthquakes. A special dense array was deployed to record the aftershocks in each case. The implications of both geological and topographical amplification are discussed in light of current code provisions. The observed geological amplifications has already influenced the code provisions. Suggestions are made to the effect that the codes should include further provisions to take the amplification due to topography into account. ?? 1991.

  17. Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis

    PubMed Central

    2014-01-01

    Background Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. Methods A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. Results The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Conclusions Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in

  18. Amplification and re-generation of LNA-modified libraries.

    PubMed

    Doessing, Holger; Hansen, Lykke H; Veedu, Rakesh N; Wengel, Jesper; Vester, Birte

    2012-11-05

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

  19. An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

    PubMed

    Thompson, Robyn E; Duncan, George; McCord, Bruce R

    2014-11-01

    A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

  20. Reverse transcription strand invasion based amplification (RT-SIBA): a method for rapid detection of influenza A and B.

    PubMed

    Eboigbodin, Kevin; Filén, Sanna; Ojalehto, Tuomas; Brummer, Mirko; Elf, Sonja; Pousi, Kirsi; Hoser, Mark

    2016-06-01

    Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.

  1. Identification of Cereulide-Producing Bacillus cereus by Nucleic Acid Chromatography and Reverse Transcription Real-Time PCR.

    PubMed

    Ueda, Shigeko; Yamaguchi, Manami; Eguchi, Kayoko; Iwase, Miki

    2016-01-01

    RNA extracts were analyzed with a nucleic acid sequence-based amplification (NASBA) - nucleic acid chromatography and a reverse transcription-quantitative PCR assay (RT-qPCR) based on the TaqMan probe for identification of cereulide-producing Bacillus cereus. All 100 emetic B. cereus strains were found to give positive results, but 50 diarrheal B. cereus strains and other bacterial species showed negative results in the NASBA-chromatography. That is, the assay could selectively identify the emetic strains among B. cereus strains. Also, the B. cereus contents of more than 10(7) cfu/ml were required for the identification of the cereulide-producing strains in this assay. In qRT-PCR assays, all 100 emetic type strains of B. cereus produced 10(2) - 10(4) copy numbers per ng of the RNA preparation, and the strains produced 10(4) copies including ones which had the high vacuolation activities of HEp-2 cells.

  2. Advancing allele group-specific amplification of the complete HLA-C gene--isolation of novel alleles from three allele groups (C*04, C*07 and C*08).

    PubMed

    Cisneros, E; Martínez-Pomar, N; Vilches, M; Martín, P; de Pablo, R; Nuñez Del Prado, N; Nieto, A; Matamoros, N; Moraru, M; Vilches, C

    2013-10-01

    A variety of strategies have been designed for sequence-based HLA typing (SBT) and for the isolation of new human leucocyte antigen (HLA) alleles, but unambiguous characterization of complete genomic sequences remains a challenge. We recently reported a simple method for the group-specific amplification (GSA) and sequencing of a full-length C*04 genomic sequence in isolation from the accompanying allele. Here we build on this strategy and present homologous methods that enable the isolation of HLA-C alleles belonging to another two allele groups. Using this approach, which can be applied to sequence-based typing in some clinical settings, we have successfully characterized three novel HLA-C alleles (C*04:128, C*07:01:01:02, and C*08:62).

  3. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  4. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  5. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of... Special Controls Guidance Document: Nucleic Acid Amplification Assay for the Detection of Enterovirus...

  6. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    PubMed

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

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

    PubMed

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

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  8. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    PubMed

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

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  9. Equipment-Free Incubation of Recombinase Polymerase Amplification Reactions Using Body Heat

    PubMed Central

    Richards-Kortum, Rebecca

    2014-01-01

    The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings. PMID:25372030

  10. Telomerase Activity Detection with Amplification-Free Single Molecule Stochastic Binding Assay.

    PubMed

    Su, Xin; Li, Zehao; Yan, Xinzhong; Wang, Lei; Zhou, Xu; Wei, Lin; Xiao, Lehui; Yu, Changyuan

    2017-03-21

    Because the elongation of telomeres has been associated with tumorigenesis, it is of great interest to develop rapid and high-confidence telomerase activity detection methods for disease diagnosis. Currently, amplification-based strategies have been extensively explored for telomerase detection in vitro and in vivo. However, amplification is typically associated with poor reproducibility and high background, which hamper further applications of the strategies, particularly for real sample assays. Here, we demonstrate a new amplification-free single molecule imaging method for telomerase activity detection in vitro based on nucleic acid stochastic binding with total internal reflection fluorescence microscopy. The dynamic stochastic binding of a short fluorescent DNA probe with a genuine target yields a distinct kinetic signature from the background noise, allowing us to identify telomerase reaction products (TRPs) at the single molecule level. A limit-of-detection as low as 0.5 fM and a dynamic range of 0.5-500 fM for TRP detection were readily achieved. With this method, telomerase extracted from cancer cells was determined with sensitivity down to 10 cells. Moreover, the length distribution of TRPs was also determined by multiple stochastic probing, which could provide deep insight into the mechanistic study of telomerase catalysis.

  11. Simultaneous multiple target detection in real-time loop-mediated isothermal amplification.

    PubMed

    Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C

    2012-08-01

    Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1-4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

  12. Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.

    PubMed

    Mathias, Jordan; Okyere, Robert; Lomidze, Levan; Gvarjaladze, David; Musier-Forsyth, Karin; Kankia, Besik

    2014-01-01

    Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.

  13. Equipment-free incubation of recombinase polymerase amplification reactions using body heat.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-01-01

    The development of isothermal amplification platforms for nucleic acid detection has the potential to increase access to molecular diagnostics in low resource settings; however, simple, low-cost methods for heating samples are required to perform reactions. In this study, we demonstrated that human body heat may be harnessed to incubate recombinase polymerase amplification (RPA) reactions for isothermal amplification of HIV-1 DNA. After measuring the temperature of mock reactions at 4 body locations, the axilla was chosen as the ideal site for comfortable, convenient incubation. Using commonly available materials, 3 methods for securing RPA reactions to the body were characterized. Finally, RPA reactions were incubated using body heat while control RPA reactions were incubated in a heat block. At room temperature, all reactions with 10 copies of HIV-1 DNA and 90% of reactions with 100 copies of HIV-1 DNA tested positive when incubated with body heat. In a cold room with an ambient temperature of 10 degrees Celsius, all reactions containing 10 copies or 100 copies of HIV-1 DNA tested positive when incubated with body heat. These results suggest that human body heat may provide an extremely low-cost solution for incubating RPA reactions in low resource settings.

  14. Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses.

    PubMed

    Moore, Matthew D; Jaykus, Lee-Ann

    2017-01-09

    Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date.

  15. Development of a Recombinase Polymerase Amplification Assay for Detection of Epidemic Human Noroviruses

    PubMed Central

    Moore, Matthew D.; Jaykus, Lee-Ann

    2017-01-01

    Human norovirus is a leading cause of viral gastroenteritis worldwide. Rapid detection could facilitate control, however widespread point-of-care testing is infrequently done due to the lack of robust and portable methods. Recombinase polymerase amplification (RPA) is a novel isothermal method which rapidly amplifies and detects nucleic acids using a simple device in near real-time. An RT-RPA assay targeting a recent epidemic human norovirus strain (GII.4 New Orleans) was developed and evaluated in this study. The assay successfully detected purified norovirus RNA from multiple patient outbreak isolates and had a limit of detection of 3.40 ± 0.20 log10 genomic copies (LGC), which is comparable to most other reported isothermal norovirus amplification methods. The assay also detected norovirus in directly boiled stool, and displayed better resistance to inhibitors than a commonly used RT-qPCR assay. The assay was specific, as it did not amplify genomes from 9 non-related enteric viruses and bacteria. The assay detected norovirus in some samples in as little as 6 min, and the entire detection process can be performed in less than 30 min. The reported RT-RPA method shows promise for sensitive point-of-care detection of epidemic human norovirus, and is the fastest human norovirus amplification method to date. PMID:28067278

  16. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    PubMed

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

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in <20 min and results in detection limits of 10 colony-forming units for methicillin-resistant Staphylococcus aureus and Salmonella enterica and 100 colony-forming units for Neisseria gonorrhoeae. The results show this method to be useful with respect to point-of-care testing and to enable simplified and miniaturized nucleic acid-based diagnostics. FigureThe combination of multiplex isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides.

  17. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

    PubMed Central

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

    2014-01-01

    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  18. Battery-triggered ultrasensitive electrochemiluminescence detection on microfluidic paper-based immunodevice based on dual-signal amplification strategy.

    PubMed

    Li, Weiping; Li, Meng; Ge, Shenguang; Yan, Mei; Huang, Jiadong; Yu, Jinghua

    2013-03-12

    Dual-signal amplification strategy for ultrasensitive electrochemiluminescence (ECL) multiplexed immunoassay on microfluidic paper-based analytical devices (μ-PADs) was demonstrated. This dual-signal amplification technique was achieved by employing graphene oxide-chitosan/gold nanoparticles (GCA) immunosensing platform and [4,4'-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid] (P-acid) functionalized nanoporous silver (P-acid/NPS) signal amplification label. For further low-cost and disposable applications, battery-triggered constant-potential ECL (+1.0V for P-acid label (vs. Ag/AgCl auxiliary electrode)) was applied on this paper-based immunodevice with the aid of a home-made voltage-tunable power device, allowing the traditional electrochemical workstation to be abandoned. We found that two tumor markers could be sequentially detected in the linear ranges of 0.003-20 and 0.001-10ngmL(-1) with the detection limits down to 1.0 and 0.8pgmL(-1), respectively, by simply reversing the connection mode on two working electrodes. The results exhibited excellent precision and high sensitivity of such immunoassay, and it also demonstrated that this battery-triggered ECL paper-based immunodevice could provide a rapid, simple and simultaneous multiplex immunoassay with high throughput, low-cost and low detection limits for point-of-care testing.

  19. Selection of key sequence-based features for prediction of essential genes in 31 diverse bacterial species

    PubMed Central

    Liu, Xiao; Wang, Bao-Jin; Xu, Luo; Tang, Hong-Ling; Xu, Guo-Qing

    2017-01-01

    Genes that are indispensable for survival are essential genes. Many features have been proposed for computational prediction of essential genes. In this paper, the least absolute shrinkage and selection operator method was used to screen key sequence-based features related to gene essentiality. To assess the effects, the selected features were used to predict the essential genes from 31 bacterial species based on a support vector machine classifier. For all 31 bacterial objects (21 Gram-negative objects and ten Gram-positive objects), the features in the three datasets were reduced from 57, 59, and 58, to 40, 37, and 38, respectively, without loss of prediction accuracy. Results showed that some features were redundant for gene essentiality, so could be eliminated from future analyses. The selected features contained more complex (or key) biological information for gene essentiality, and could be of use in related research projects, such as gene prediction, synthetic biology, and drug design. PMID:28358836

  20. Sequence-Based Identification of Mycobacterium Species Using the MicroSeq 500 16S rDNA Bacterial Identification System

    PubMed Central

    Patel, Jean Baldus; Leonard, Debra G. B.; Pan, Xai; Musser, James M.; Berman, Richard E.; Nachamkin, Irving

    2000-01-01

    We evaluated the MicroSeq 500 16S rDNA Bacterial Sequencing Kit (PE Applied Biosystems), a 500-bp sequence-based identification system, for its ability to identify clinical Mycobacterium isolates. The organism identity was determined by comparing the 16S rDNA sequence to the MicroSeq database, which consists primarily of type strain sequences. A total of 113 isolates (18 different species), previously recovered and identified by routine methods from two clinical laboratories, were analyzed by the MicroSeq method. Isolates with discordant results were analyzed by hsp65 gene sequence analysis and in some cases repeat phenotypic identification, AccuProbe rRNA hybridization (Gen-Probe, Inc., San Diego, Calif.), or high-performance liquid chromatography of mycolic acids. For 93 (82%) isolates, the MicroSeq identity was concordant with the previously reported identity. For 18 (16%) isolates, the original identification was discordant with the MicroSeq identification. Of the 18 discrepant isolates, 7 (six unique sequences) were originally misidentified by phenotypic analysis or the AccuProbe assay but were correctly identified by the MicroSeq assay. Of the 18 discrepant isolates, 11 (seven unique sequences) were unusual species that were difficult to identify by phenotypic methods and, in all but one case, by molecular methods. The remaining two isolates (2%) failed definitive phenotypic identification, but the MicroSeq assay was able to definitively identify one of these isolates. The MicroSeq identification system is an accurate and rapid method for the identification of Mycobacterium spp. PMID:10618095

  1. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  2. Backward Raman amplification in the long-wavelength infrared

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

    The wealth of work in backward Raman amplification in plasma has focused on the extreme intensity limit; however, backward Raman amplification may also provide an effective and practical mechanism for generating intense, broad bandwidth, long-wavelength infrared radiation (LWIR). An electromagnetic simulation coupled with a relativistic cold fluid plasma model is used to demonstrate the generation of picosecond pulses at a wavelength of 10 μm with terawatt powers through backward Raman amplification. The effects of collisional damping, Landau damping, pump depletion, and wave breaking are examined, as well as the resulting design considerations for an LWIR Raman amplifier.

  3. Gene and library synthesis without amplification: polymerase step reaction (PSR).

    PubMed

    Lee, Zhuo-Bin; Firnhaber, Christopher; Clarke, Jesse; DeDecker, Brian S

    2015-09-01

    Current gene synthesis methods often incorporate a PCR amplification step in order to yield final material sufficient for resolution from multiple off-products. These amplification steps can cause stochastic sampling effects that propagate errors in gene synthesis or decrease variability when applied to the construction of randomized libraries. We have developed a simple DNA polymerase-based gene synthesis reaction, polymerase step reaction (PSR), that assembles DNA oligonucleotides in a unidirectional fashion without the need for amplification. We demonstrate that PSR is efficient, with little off-product production, no detectable error propagation, and maximized variability in the synthesis of a phage display library.

  4. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  5. Optical amplification and reshaping based on the Peregrine rogue wave.

    PubMed

    Wang, Yan; Song, Lijun; Li, Lu

    2016-09-10

    Based on the characteristics of the Peregrine rogue wave, the amplification and the reshaping of solitons are investigated. The numerical results show that the amplification and the reshaping of solitons can be realized by injecting a continuous wave (CW) and filtering the CW at suitable positions. The combination of a continuous-wave pump and a spectral filter placed suitably in fiber plays the role of the amplifier, which can be used to long-haul the transmission of solitons. As an example, a long-haul transmission with four amplification periods is demonstrated.

  6. Fluorescence amplification by electrochemically deposited silver nanowires with fractal architecture.

    PubMed

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

    2007-10-10

    Electrochemically deposited silver structures with nanowires 50-100 nm in diameter show high fluorescence amplification and strongly reduced fluorescence lifetimes. Both quantities depend on the structure thickness. With increasing thickness the fluorescence amplification proportionally increases and the fluorescence lifetime decreases. This thickness dependence is caused by fluorophore interaction with a system of plasmon excitations in coupled nanowires extending over micrometer size regions. Thus the amplification is attributed to a combination of extended structure area and strong plasmonic coupling between nanowires which also help to radiatively scatter the fluorescence emission.

  7. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    PubMed

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

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  8. Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care.

    PubMed

    Lillis, Lorraine; Siverson, Joshua; Lee, Arthur; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Lehman, Dara A; Boyle, David S

    2016-04-01

    Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in under 20 min without the need for complex instrumentation. These properties enable its potential use in resource limited settings. However, there are concerns that deviations from the manufacturer's protocol and/or storage conditions could influence its performance in low resource settings. RPA amplification relies upon viscous crowding agents for optimal nucleic acid amplification, and thus an interval mixing step after 3-6 min of incubation is recommended to distribute amplicons and improve performance. In this study we used a HIV-1 RPA assay to evaluate the effects of this mixing step on assay performance. A lack of mixing led to a longer time to amplification and inferior detection signal, compromising the sensitivity of the assay. However lowering the assay volume from 50 μL to 5 μL showed similar sensitivity with or without mixing. We present the first peer-reviewed study that assesses long term stability of RPA reagents without a cold chain. Reagents stored at -20 °C, and 25 °C for up to 12 weeks were able to detect 10 HIV-1 DNA copies. Reagents stored at 45 °C for up to 3 weeks were able to detect 10 HIV-1 DNA copies, with reduced sensitivity only after >3 weeks at 45 °C. Together our results show that reducing reaction volumes bypassed the need for the mixing step and that RPA reagents were stable even when stored for 3 weeks at very high temperatures.

  9. A general solution for opening double-stranded DNA for isothermal amplification

    PubMed Central

    Chen, Gangyi; Dong, Juan; Yuan, Yi; Li, Na; Huang, Xin; Cui, Xin; Tang, Zhuo

    2016-01-01

    Nucleic acid amplification is the core technology of molecular biology and genetic engineering. Various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). However, most of these methods can only detect single stranded nucleic acid. Herein, we put forward a simple solution for opening double-stranded DNA for isothermal detection methods. The strategy employs recombination protein from E. coli (RecA) to form nucleoprotein complex with single-stranded DNA, which could scan double-stranded template for homologous sites. Then, the nucleoprotein can invade the double-stranded template to form heteroduplex in the presence of ATP, resulting in the strand exchange. The ATP regeneration system could be eliminated by using high concentration of ATP, and the 3′-OH terminal of the invasion strand can be recognized by other DNA modifying enzymes such as DNA polymerase or DNA ligase. Moreover, dATP was found to be a better cofactor for RecA, which make the system more compatible to DNA polymerase. The method described here is a general solution to open dsDNA, serving as a platform to develop more isothermal nucleic acids detection methods for real DNA samples based on it. PMID:27687498

  10. KASER: Knowledge Amplification by Structured Expert Randomization.

    PubMed

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

    2004-12-01

    In this paper and attached video, we present a third-generation expert system named Knowledge Amplification by Structured Expert Randomization (KASER) for which a patent has been filed by the U.S. Navy's SPAWAR Systems Center, San Diego, CA (SSC SD). KASER is a creative expert system. It is capable of deductive, inductive, and mixed derivations. Its qualitative creativity is realized by using a tree-search mechanism. The system achieves creative reasoning by using a declarative representation of knowledge consisting of object trees and inheritance. KASER computes with words and phrases. It possesses a capability for metaphor-based explanations. This capability is useful in explaining its creative suggestions and serves to augment the capabilities provided by the explanation subsystems of conventional expert systems. KASER also exhibits an accelerated capability to learn. However, this capability depends on the particulars of the selected application domain. For example, application domains such as the game of chess exhibit a high degree of geometric symmetry. Conversely, application domains such as the game of craps played with two dice exhibit no predictable pattern, unless the dice are loaded. More generally, we say that domains whose informative content can be compressed to a significant degree without loss (or with relatively little loss) are symmetric. Incompressible domains are said to be asymmetric or random. The measure of symmetry plus the measure of randomness must always sum to unity.

  11. Cyclic Amplification of Prion Protein Misfolding

    PubMed Central

    Barria, Marcelo A; Gonzalez-Romero, Dennisse; Soto, Claudio

    2014-01-01

    Protein Misfolfing Cyclic amplification (PMCA) is a technique that take advantage of the nucleation-dependent prion replication process to accelerate the conversion of PrPC into PrPSc in the test tube. PMCA uses ultrasound waves to fragment the PrPSc polymers, increasing the amount of seeds present in the infected sample without affecting their ability to act as conversion nucleus. Over the past 5 years PMCA has became an invaluable technique to study diverse aspects of prions. The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the cellular factors involved in prion propagation, the intriguing phenomena of prion strains and species barriers, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. In this article we describe a detailed protocol of the PMCA technique, highlighting some of the important technical aspects to obtain a successful and reproducible application of the technology. PMID:22528092

  12. Small Sample Whole-Genome Amplification

    SciTech Connect

    Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P; Christian, A T

    2005-09-20

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  13. Small sample whole-genome amplification

    NASA Astrophysics Data System (ADS)

    Hara, Christine; Nguyen, Christine; Wheeler, Elizabeth; Sorensen, Karen; Arroyo, Erin; Vrankovich, Greg; Christian, Allen

    2005-11-01

    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.

  14. Magnetic field amplification in turbulent astrophysical plasmas

    NASA Astrophysics Data System (ADS)

    Federrath, Christoph

    2016-12-01

    Magnetic fields play an important role in astrophysical accretion discs and in the interstellar and intergalactic medium. They drive jets, suppress fragmentation in star-forming clouds and can have a significant impact on the accretion rate of stars. However, the exact amplification mechanisms of cosmic magnetic fields remain relatively poorly understood. Here, I start by reviewing recent advances in the numerical and theoretical modelling of the turbulent dynamo, which may explain the origin of galactic and intergalactic 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 simulations from which I determine the growth of the turbulent (un-ordered) magnetic field component ( turb$ ) in the presence of weak and strong guide fields ( 0$ ). I vary 0$ over five orders of magnitude and find that the dependence of turb$ on 0$ is relatively weak, and can be explained with a simple theoretical model in which the turbulence provides the energy to amplify turb$ . Finally, I discuss some important implications of magnetic fields for the structure of accretion discs, the launching of jets and the star-formation rate of interstellar clouds.

  15. Macroscopic Velocity Amplification in Stacked Disks

    NASA Astrophysics Data System (ADS)

    Murthy, Srividya; White, Gary

    2015-04-01

    When a small sphere rests atop a larger sphere (for example, a basketball with a tennis ball balanced on top), and both are released from a height, the resulting ``velocity amplification'' of the small sphere when the pair rebound from a hard floor, is a staple of the physics demonstration toolkit--usually impressive, sometimes dangerous. While this phenomenon has been studied in the literature in some detail, we set out to explore this effect by constructing a device involving stacked disks falling in a plane, fashioned after an online design by Wayne Peterson of Brigham Young University. When two disks, stacked edge to edge atop one another and confined to a vertical plane, are dropped, the top disk rebounds to a much greater height than it started from, as expected. In this talk, we report on experiments conducted by dropping the disks and recording the heights to which they rise on rebound, and the comparison of these results with our theoretical predictions and computer simulations. Frances E. Walker Fellowship.

  16. Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field.

    PubMed

    Dickinson, Matt

    2015-01-01

    Loop-mediated isothermal amplification (LAMP) is a nucleic acid-based detection method with many applications. This chapter details its use for detection of phytoplasma diseases, combining a rapid 2-min DNA extraction method with real-time LAMP product detection such that the entire procedure can be undertaken and completed in the field within 40 min. Furthermore, the assays include an anneal curve validation step to guard against false positives and an assay for host plant DNA to guard against false negatives.

  17. Highly simplified lateral flow-based nucleic acid sample preparation and passive fluid flow control

    SciTech Connect

    Cary, Robert E.

    2015-12-08

    Highly simplified lateral flow chromatographic nucleic acid sample preparation methods, devices, and integrated systems are provided for the efficient concentration of trace samples and the removal of nucleic acid amplification inhibitors. Methods for capturing and reducing inhibitors of nucleic acid amplification reactions, such as humic acid, using polyvinylpyrrolidone treated elements of the lateral flow device are also provided. Further provided are passive fluid control methods and systems for use in lateral flow assays.

  18. Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens.

    PubMed

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

    2011-06-01

    The preparation and use of a new class of signal amplification label, quantum dot (QD) layer-by-layer (LBL) assembled polystyrene microsphere composite, for amplified ultrasensitive electronic detection of uropathogen-specific DNA sequences is described. The target DNA is sandwiched between the capture probes immobilized on the magnetic beads and the signaling probes conjugated to the QD LBL assembled polystyrene beads. Because of the dramatic signal amplification by the numerous QDs involved in each single DNA binding event, subfemtomolar level detection of uropathogen-specific DNA sequences is achieved, which makes our strategy among the most sensitive electronic approach for nucleic acid-based monitoring of pathogens. Our signal amplified detection scheme could be readily expanded to monitor other important biomolecules (e.g., proteins, peptides, amino acids, cells, etc.) in ultralow levels and thus holds great potential for early diagnosis of disease biomarkers.

  19. AFB (Acid-Fast Bacillus) Smear and Culture

    MedlinePlus

    ... Fast Bacillus Smear and Culture and Sensitivity; Mycobacteria tuberculosis Nucleic Acid Amplification Test Related tests: TB Screening ... is most commonly used to identify an active tuberculosis (TB) infection caused by the most medically important ...

  20. Nonlinear Zel'dovich Effect: Parametric Amplification from Medium Rotation

    NASA Astrophysics Data System (ADS)

    Faccio, Daniele; Wright, Ewan M.

    2017-03-01

    The interaction of light with rotating media has attracted recent interest for both fundamental and applied studies including rotational Doppler shift measurements. It is also possible to obtain amplification through the scattering of light with orbital angular momentum from a rotating and absorbing cylinder, as proposed by Zel'dovich more than forty years ago. This amplification mechanism has never been observed experimentally yet has connections to other fields such as Penrose superradiance in rotating black holes. Here we propose a nonlinear optics system whereby incident light carrying orbital angular momentum drives parametric interaction in a rotating medium. The crystal rotation is shown to take the phase-mismatched parametric interaction with negligible energy exchange at zero rotation to amplification for sufficiently large rotation rates. The amplification is shown to result from breaking of anti-P T symmetry induced by the medium rotation.

  1. Loop-mediated isothermal amplification of single pollen grains.

    PubMed

    Bektaş, Ali; Chapela, Ignacio

    2014-08-01

    The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification 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 pollen 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 pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field-based, high-throughput analysis.

  2. Three-dimensional Simulation of Backward Raman Amplification

    SciTech Connect

    A.A. Balakin; G.M. Fraiman; N.J. Fisch

    2005-11-12

    Three-dimensional (3-D) simulations for the Backward Raman Amplification (BRA) are presented. The images illustrate the effects of pump depletion, pulse diffraction, non-homogeneous plasma density, and plasma ionization.

  3. Amplification of surface temperature trends and variability in thetropical atmosphere

    SciTech Connect

    Santer, B.D.; Wigley, T.M.L.; Mears, C.; Wentz, F.J.; Klein,S.A.; Seidel, D.J.; Taylor, K.E.; Thorne, P.W.; Wehner, M.F.; Gleckler,P.J.; Boyle, J.S.; Collins, W.D.; Dixon, K.W.; Doutriaux, C.; Free, M.; Fu, Q.; Hansen, J.E.; Jones, G.S.; Ruedy, R.; Karl, T.R.; Lanzante, J.R.; Meehl, G.A.; Ramaswamy, V.; Russell, G.; Schmidt, G.A.

    2005-08-11

    The month-to-month variability of tropical temperatures is larger in the troposphere than at the Earth's surface. This amplification behavior is similar in a range of observations and climate model simulations, and is consistent with basic theory. On multi-decadal timescales, tropospheric amplification of surface warming is a robust feature of model simulations, but occurs in only one observational dataset. Other observations show weak or even negative amplification. These results suggest that either different physical mechanisms control amplification processes on monthly and decadal timescales, and models fail to capture such behavior, or (more plausibly) that residual errors in several observational datasets used here affect their representation of long-term trends.

  4. Preliminary Lidar Experiment to Study the Backscatter Amplification

    NASA Astrophysics Data System (ADS)

    Razenkov, Igor A.; Banakh, Victor A.

    2016-06-01

    Long-term continuous measurements for detection relative backscatter amplification on a horizontal path of 2 km long are performed by using a specific micro pulse lidar. The laser beam path is limited by a solid obstacle. The lidar is located next to an ultrasonic anemometer that measures 3D wind velocity and temperature; the laser spot on the obstacle is observed by using a telephoto lens. The results showed that the backscatter amplification has a clear diurnal variation. Moreover, the backscatter amplification was completely absent in the morning and evening under neutral stratification in the atmospheric surface layer. At night and in the daytime there was a significant increase of the backscatter amplification coefficient.

  5. Centrosome Amplification Is Sufficient to Promote Spontaneous Tumorigenesis in Mammals.

    PubMed

    Levine, Michelle S; Bakker, Bjorn; Boeckx, Bram; Moyett, Julia; Lu, James; Vitre, Benjamin; Spierings, Diana C; Lansdorp, Peter M; Cleveland, Don W; Lambrechts, Diether; Foijer, Floris; Holland, Andrew J

    2017-02-06

    Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.

  6. Loop-Mediated Isothermal Amplification (LAMP) Signature Identification Software

    SciTech Connect

    Torres, C.

    2009-03-17

    This is an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.

  7. Optical pulse synthesis using brillouin selective sideband amplification

    NASA Technical Reports Server (NTRS)

    Yao, X. Steve (Inventor)

    2002-01-01

    Techniques for producing optical pulses based on Brillouin selective sideband amplification by using a common modulation control signal to modulate both a signal beam to produce multiple sideband signals and a single pump beam to produce multiple pump beams.

  8. Backward Raman amplification of broad-band pulses

    NASA Astrophysics Data System (ADS)

    Balakin, A. A.; Dodin, I. Y.; Fraiman, G. M.; Fisch, N. J.

    2016-08-01

    A reduced fluid model of Raman backscattering is proposed that describes backward Raman amplification (BRA) of pulses with duration τ0 comparable to or even smaller than the plasma period 2 π/ωp . At such a small τ0, a seed pulse can be amplified even if it has the same frequency as the pump (which is technologically advantageous), as opposed to that satisfying the Raman resonance condition. Using our theoretical model, we numerically calculate the BRA efficiency for such pulses as a function of τ0 and show that it remains reasonably high up to τ0≈2 π/ωp . We also show that using short seed pulses in BRA makes the amplification less sensitive to quasistatic inhomogeneities of the plasma density. Amplification can persist even when the density perturbations are large enough to violate the commonly known condition of resonant amplification.

  9. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    PubMed

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

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

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

    PubMed Central

    2016-01-01

    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. PMID:26900709

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

    PubMed

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

  12. Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

    PubMed Central

    Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

    2012-01-01

    Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

  13. Low-noise preamplifier for multistage photorefractive image amplification

    NASA Astrophysics Data System (ADS)

    Breugnot, S.; Rajbenbach, H.; Defour, M.; Huignard, J.-P.

    1995-07-01

    We present a two-beam coupling configuration in photorefractive BaTiO3 that provides a low-noise amplification of the signal to be detected. A two-wave mixing gain of 100 is reached, in conjunction with very low beam fanning background in the signal direction. The extensions of this configuration to photorefractive heterodyne detection and to multistage image amplification are theoretically and experimentally studied.

  14. Simulation study of electron response amplification in coherent electron cooling

    SciTech Connect

    Hao Y.; Litvinenko, V.N.

    2012-05-20

    In Coherent Electron Cooling (CEC), it is essential to study the amplification of electron response to a single ion in the FEL process, in order to proper align the electron beam and the ion beam in the kicker to maximize the cooling effect. In this paper, we use Genesis to simulate the amplified electron beam response of single ion in FEL amplification process, which acts as Green's function of the FEL amplifier.

  15. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2000-01-01

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  16. Detection of biological molecules using chemical amplification and optical sensors

    DOEpatents

    Van Antwerp, William Peter; Mastrototaro, John Joseph

    2004-10-12

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  17. Detection of biological molecules using chemical amplification and optical sensors

    SciTech Connect

    Antwerp, W.P. van; Mastrototaro, J.J.

    2000-01-04

    Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal.

  18. Host Amplification in a Dithioacetal-Based Dynamic Covalent Library.

    PubMed

    Orrillo, A Gastón; Escalante, Andrea M; Furlan, Ricardo L E

    2017-03-06

    Molecular amplification in a dithioacetal-based dynamic library is described for the first time. The homatropine induced selection, amplification, and isolation of one cyclophane host demonstrates the utility of dithioacetal exchange for preparing responsive dynamic libraries. Nuclear magnetic resonance and isothermal titration calorimetry analysis suggest that the amplified macrocycle forms a 1:1 complex with the template. This is the first report about a host/guest system involving a dithioacetal cyclophane.

  19. Amplification of ultra-short laser pulses via resonant backward Raman amplification in plasma

    NASA Astrophysics Data System (ADS)

    Mishra, S. K.; Andreev, A.

    2016-08-01

    In this paper, we have examined the possibility of using resonant backward Raman amplification (BRA) as an efficient mechanism in amplifying the low intensity ultra-short ( ≤ fs ) pulses using plasma as intermediate amplifying medium; such pulses are anticipated to get produced in the form of the secondary sources at ALPS (Attosecond Light Pulse Source) center of ELI (Extreme Light Infrastructure). In preliminary assessment of the scheme, the analytical expressions for the pump/seed laser pulses and plasma characteristic features are obtained which concisely describe the parameter regime of resonant BRA applicability in achieving significant amplification. The consistency of the scheme in the context of ELI-ALPS sources has been validated through particle in cell (PIC) simulations. The peak intensity of the amplified seed pulse predicted via simulation results is found in reasonable agreement with the analytical estimates. Utilizing these analytical expressions as a basis in perspective of ELI-ALPS parameter access, a specific example displaying the key plasma and laser parameters for amplifying weak seed pulse has been configured; the limitations and conceivable remedies in resonant BRA implementation have also been highlighted.

  20. Role of Atmospheric Transport on the Arctic Amplification: Adjusting Role

    NASA Astrophysics Data System (ADS)

    KUG, J.; Yim, B.; Jin, F.

    2013-12-01

    It is controversial whether the atmospheric transport plays a role in arctic amplification. Recently, Hwang et al. (2011) showed that the magnitude of the arctic amplification is negatively correlated with anomalous poleward atmospheric transport. That is, when the arctic amplification is strong (weak), the atmospheric transport plays a negative (positive) role in the arctic amplification. In this study, it is discussed what is a physical mechanism to determine the role of atmospheric transport and relation with the arctic amplification. Here, we suggest adjusting roles of atmospheric transport. The strength of local feedback over the Arctic determines zonal wind changes. The zonal wind changes are determined by two factors. The first one is polar cap cooling, and second is surface warming. They play opposite roles. So, there will be two different zonal wind responses in high-latitude to the greenhouse warming. Depending on the zonal wind response, the atmospheric transport can play a different role because the zonal wind changes can organize synoptic eddy feedbacks including heat flux, which largely contributes to poleward energy transport. We show here that when polar cap cooling is strong, and surface warming over Arctic is relatively weak, the Jet stream tends to be shifted poleward, so it leads to poleward atmospheric transport. On the other hand, when the surface warming is too strong, it lead to southward shift of Jet stream and equatorward atmospheric transport, which paly a negative role in the Arctic amplification.

  1. Mechanical amplification by hair cells in the semicircular canals.

    PubMed

    Rabbitt, Richard D; Boyle, Richard; Highstein, Stephen M

    2010-02-23

    Sensory hair cells are the essential mechanotransducers of the inner ear, responsible not only for the transduction of sound and motion stimuli but also, remarkably, for nanomechanical amplification of sensory stimuli. Here we show that semicircular canal hair cells generate a mechanical nonlinearity in vivo that increases sensitivity to angular motion by amplification at low stimulus strengths. Sensitivity at high stimulus strengths is linear and shows no evidence of amplification. Results suggest that the mechanical work done by hair cells contributes approximately 97 zJ/cell of amplification per stimulus cycle, improving sensitivity to angular velocity stimuli below approximately 5 degrees /s (0.3-Hz sinusoidal motion). We further show that mechanical amplification can be inhibited by the brain via activation of efferent synaptic contacts on hair cells. The experimental model was the oyster toadfish, Opsanus tau. Physiological manifestation of mechanical amplification and efferent control in a teleost vestibular organ suggests the active motor process in sensory hair cells is ancestral. The biophysical basis of the motor(s) remains hypothetical, but a key discriminating question may involve how changes in somatic electrical impedance evoked by efferent synaptic action alter function of the motor(s).

  2. Empirical evidence for acceleration-dependent amplification factors

    USGS Publications Warehouse

    Borcherdt, R.D.

    2002-01-01

    Site-specific amplification factors, Fa and Fv, used in current U.S. building codes decrease with increasing base acceleration level as implied by the Loma Prieta earthquake at 0.1g and extrapolated using numerical models and laboratory results. The Northridge earthquake recordings of 17 January 1994 and subsequent geotechnical data permit empirical estimates of amplification at base acceleration levels up to 0.5g. Distance measures and normalization procedures used to infer amplification ratios from soil-rock pairs in predetermined azimuth-distance bins significantly influence the dependence of amplification estimates on base acceleration. Factors inferred using a hypocentral distance norm do not show a statistically significant dependence on base acceleration. Factors inferred using norms implied by the attenuation functions of Abrahamson and Silva show a statistically significant decrease with increasing base acceleration. The decrease is statistically more significant for stiff clay and sandy soil (site class D) sites than for stiffer sites underlain by gravely soils and soft rock (site class C). The decrease in amplification with increasing base acceleration is more pronounced for the short-period amplification factor, Fa, than for the midperiod factor, Fv.

  3. Population diversity of ammonium oxidizers investigated by specific PCR amplification

    USGS Publications Warehouse

    Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

    1997-01-01

    The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

  4. Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

    PubMed

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

    2016-12-15

    To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

  5. Ultrasensitive DNA detection by cycle isothermal amplification based on nicking endonuclease and its application to logic gates.

    PubMed

    Li, Xuemei; Ding, Tianrong; Sun, Li; Mao, Changming

    2011-12-15

    In recent years, an intense interest has grown in the DNA logic gates having high potential for computation at literally the "nano-size" level. A limitation of traditional DNA logic gates is that each target strand hybridizes with only a single copy of the probe. This 1:1 hybridization radio limits the gain of the approach and thus its sensitivity. The exponential amplification of nucleic acids has become a core technology in medical diagnostics and has been widely used for the construction of DNA sensor, DNA nanomachine and DNA sequencing. It would be of great interest to develop DNA-based logic systems with exponential amplification for the output signal. In the present study, a series of three-input DNA logic gates with the cycle isothermal amplification based on nicking endonuclease (NEase) are designed. Very low concentrations of the analytes were sufficient to initiate an autocatalytic cascade, achieving a significant improvement of the detection limit, 100-fold improvement compared to the non-autocatalytic system. This was achieved by engineering a simple and flexible biological circuit designed to initiate a cascade of events to detect and amplify a specific DNA sequence. This procedure has the potential to greatly simplify the logic operation because amplification can be performed in "one-pot".

  6. From Interface to Solution: Integrating Immunoassay with Netlike Rolling Circle Amplification for Ultrasensitive Detection of Tumor Biomarker

    PubMed Central

    Feng, Chang; Bo, Bing; Mao, Xiaoxia; Shi, Hai; Zhu, Xiaoli; Li, Genxi

    2017-01-01

    Both the 3D solution and the 2D interface play important roles in bioanalysis. For the former, reactions can be carried out adequately; while for the latter, interfering substance can be eliminated simply through wash. It is a challenge to integrate the advantages of solution-based assays and the interface-based assays. Here, we report an immuno-NRCA (netlike rolling circle amplification) strategy, which integrates immunoassay with NRCA for the ultrasensitive detection of tumor biomarker, by taking the assay of a tumour marker as an example. In this strategy, immunoreactions occur on interface, while the target-induced signal amplification can be completed totally in solution. As a result, this system has the merits of both solution- and interface-based assays. The whole procedure of this novel strategy is similar to the conventional ELISA, inheriting the usability. But in comparison with ELISA, the performance is greatly improved. The detection limit can be lowered to 5.5 fg/L, making it possible to detect the target tumour marker in one drop of blood. Also, in comparison with established immuno-PCR method, which integrates immunoassay with the commonly used nucleic acid amplification approach, this system has no requirement for thermal cycler owing to the isothermal amplification, and it has the ability to retain the immunoreactivities. So, the new immunoassay method proposed in this study may have more feasible applications in the future. PMID:28042314

  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

    PubMed Central

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

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC. PMID:27749897

  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.

    PubMed

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

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

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

    PubMed

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

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  10. Amplification of prolamin storage protein genes in different subfamilies of the Poaceae.

    PubMed

    Xu, Jian-Hong; Messing, Joachim

    2009-11-01

    Prolamins are seed storage proteins in cereals and represent an important source of essential amino acids for feed and food. Genes encoding these proteins resulted from dispersed and tandem amplification. While previous studies have concentrated on protein sequences from different grass species, we now can add a new perspective to their relationships by asking how their genes are shared by ancestry and copied in different lineages of the same family of species. These differences are derived from alignment of chromosomal regions, where collinearity is used to identify prolamin genes in syntenic positions, also called orthologous gene copies. New or paralogous gene copies are inserted in tandem or new locations of the same genome. More importantly, one can detect the loss of older genes. We analyzed chromosomal intervals containing prolamin genes from rice, sorghum, wheat, barley, and Brachypodium, representing different subfamilies of the Poaceae. The Poaceae commonly known as the grasses includes three major subfamilies, the Ehrhartoideae (rice), Pooideae (wheat, barley, and Brachypodium), and Panicoideae (millets, maize, sorghum, and switchgrass). Based on chromosomal position and sequence divergence, it becomes possible to infer the order of gene amplification events. Furthermore, the loss of older genes in different subfamilies seems to permit a faster pace of divergence of paralogous genes. Change in protein structure affects their physical properties, subcellular location, and amino acid composition. On the other hand, regulatory sequence elements and corresponding transcriptional activators of new gene copies are more conserved than coding sequences, consistent with the tissue-specific expression of these genes.

  11. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  12. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    PubMed Central

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

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification. PMID:26729209

  13. Single-use, electricity-free amplification device for detection of HIV-1.

    PubMed

    Curtis, Kelly A; Rudolph, Donna L; Morrison, Daphne; Guelig, Dylan; Diesburg, Steven; McAdams, David; Burton, Robert A; LaBarre, Paul; Owen, Michele

    2016-11-01

    Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

  14. Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Litopenaeus vannamei by ramification amplification assay.

    PubMed

    Teng, Ping-Hua; Lee, Pei-Yu; Lee, Fu-Chun; Chien, Hung-Wen; Chen, Man-Shu; Sung, Ping-Feng; Su, Chen; Ou, Bor-Rung

    2006-12-14

    Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is a single-stranded DNA virus that causes developmental and growth abnormalities in Pacific white shrimp Litopenaeus vannamei (also known as Penaeus vannamei). Nucleic acid based methods such as in situ hybridization (ISH) and PCR have been commonly used for IHHNV detection. Ramification amplification (RAM), an isothermal nucleic acid amplification approach, was used in this study to detect IHHNV in L. vannamei. RAM offers many advantages over PCR, including simple procedures and short detection time, and is labor-saving and cost-effective. RAM exponentially amplifies a circular oligonucleotide amplicon (C probe) after a target-specific ligation step through sequential primer extension and strand displacement processes. The conditions of an IHHNV RAM assay were optimized using artificial templates and targets prior to application. Using DNA of IHHNV-infected L. vannamei as targets, results revealed that RAM amplified target DNA with similar sensitivity as PCR. RAM offers competitive levels of speed, simplicity and sensitivity among various pathogen diagnostic methods.

  15. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

    NASA Astrophysics Data System (ADS)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  16. HLA-C locus allelic dropout in Sanger sequence-based typing due to intronic single nucleotide polymorphism.

    PubMed

    Cheng, Christopher; Kashi, Zahra Mehdizadeh; Martin, Russell; Woodruff, Gillian; Dinauer, David; Agostini, Tina

    2014-12-01

    We report a novel HLA-C allele that was identified during routine HLA typing using sequence-based methods. The patient was initially typed as a C*06:02, 06:04 with two nucleotide mismatches in exon 3, (C to T and T to G changes) which would have resulted in a non-synonymous mutation of a leucine residue being replaced with tryptophan. Further resolution of the patient's type by using sequence-specific primers (SSP) revealed that the companion allele to C*06:02 was a novel C*17:01. Confirmation of the existence of the new allele was performed across multiple platforms: Sanger sequencing, SSP, and Next Generation Sequencing (NGS) on the original sample and allele-specific clones for the entire HLA-C locus. The investigation revealed a single nucleotide mismatch within the Sanger sequencing primer binding site in intron 3. The mutation caused the initial C*17 dropout in exons 2 and 3. Further analysis of the Sanger and NGS data revealed that the C*17 had two additional unique positions in introns 2 and 7. The companion C*06:02 allele also possessed a novel position at intron 3. On August 31, 2013, the WHO nomenclature committee officially named the novel C*17:01 allele sequence as C*17:01:01:03 and the novel C*06:02 allele sequence as C*06:02:01:03.

  17. Ligation-mediated PCR, a fast and reliable technique for insertion sequence-based typing of Xanthomonas citri pv. citri.

    PubMed

    Ngoc, Lan Bui Thi; Vernière, Christian; Belasque, José Júnior; Vital, Karine; Boutry, Sébastien; Gagnevin, Lionel; Pruvost, Olivier

    2008-11-01

    Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.

  18. Reanalysis of sequence-based HLA-A, -B and -Cw typings: how ambiguous is today's SBT typing tomorrow.

    PubMed

    Voorter, C E M; Mulkers, E; Liebelt, P; Sleyster, E; van den Berg-Loonen, E M

    2007-11-01

    The permanently increasing number of human leukocyte antigen (HLA)-alleles and the growing list of ambiguities require continuous updating of high-resolution HLA typing results. Two different kinds of ambiguities exist: the first, when two or more allele combinations have identical heterozygous sequences, and the second, when differences are located outside the analyzed region. The number of HLA-A, B and C alleles recognized in 1999 was almost tripled in 2006. Two hundred individuals, sequence-based typing (SBT) typed in the period from 1999 to 2002, were reanalyzed using the 2006 database. A final allele typing result of at least four digits was obtained for HLA-A, -B and -C by heterozygous sequencing of exons 2 and 3 and, if necessary, additional exons and/or allele-specific sequencing. Storage of the individual sequences in a specially developed database enabled reanalysis with all present and future HLA releases. In the 5-year period HLA-A, -B and -C typing results became ambiguous in 37%, 46% and 41% of the cases. Most were because of differences outside the analyzed region; ambiguities because of different allele combinations with identical heterozygous sequences were present in 7%, 8% and 13% of the HLA-A, -B and -C typings. These results indicate that within 5 years, approximately half of the HLA SBT typings become ambiguous.

  19. Nosocomial Legionnaires' disease caused by Legionella pneumophila serogroup 6: implication of the sequence-based typing method (SBT).

    PubMed

    Fendukly, Faiz; Bernander, Sverker; Hanson, Hanna-Stina

    2007-01-01

    Sequence-based typing (SBT) was used to determine the allelic profiles of 3 sporadic clinical isolates as well as 7 environmental isolates of Legionella pneumophila serogroup 6, isolated at the Karolinska Hospital during 2004. The clinical isolates were cultured from patients with nosocomial Legionnaires' disease (LD), while the environmental isolates were cultured from potable water sources of the hospital wards in the close vicinity of the 3 patients being investigated. The genes sequenced for the construction of the SBT profile included flaA, pilE, asd, mip, mompS and proA, in this pre-determined order and the allelic profile of the 10 isolates was identical (3, 13, 1, 28, 14, 9). Furthermore, 2 of the isolates, 1 clinical and 1 environmental, were analysed using the amplified fragment length polymorphism analysis (AFLP). The AFLP genotype of both isolates was congruent. Eight of 9 control L. pneumophila serogroup 6 isolates had the same SBT profile as the study isolates. We conclude that the environmental strain isolated from our hospital's drinking water is indistinguishable genotypically from the 3 clinical isolates of Legionella. However, this genotype of L. pneumophila is geographically widespread. Thus, results of genotyping must be evaluated in conjunction with the clinical and epidemiological data.

  20. Sequence-based Methods in Human Microbial Ecology: A The 2nd HumanGenome Comes of Age

    SciTech Connect

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

    2005-06-01

    Ecologists studying microbial life in the environment have recognized the enormous complexity of microbial diversity for more than a decade (Whitman et al. 1998). The development of a variety of culture-independent methods, many of them coupled with high-throughput DNA sequencing, has allowed this diversity to be explored in ever greater detail (Handelsman 2004; Harris et al. 2004; Hugenholtz et al. 1998; Moreira and Lopez-Garcia 2002; Rappe and Giovannoni 2003). Despite the widespread application of these new techniques to the characterization of uncultivated microbes and microbial communities in the environment, their application to human health and disease has lagged behind. Because these techniques now allow not only cataloging of microbial diversity, but also insight into microbial functions, it is time for clinical microbiologists to apply these tools to the microbial communities that abound on and within us, in what has been aptly called ''the second Human Genome Project'' (Relman and Falkow 2001). In this review we will discuss the sequence-based methods for microbial analysis that are currently available and their application to identify novel human pathogens, improve diagnosis and treatment of known infectious diseases, and finally to advance understanding of our relationship with microbial communities that normally reside in and on the human body.

  1. Sequence-based definition of eight short tandem repeat loci located within the HLA-region in an Austrian population.

    PubMed

    Dauber, Eva-Maria; Wenda, Sabine; Schwartz-Jungl, Elisabeth Maria; Glock, Barbara; Mayr, Wolfgang R

    2015-01-01

    Sequenced allelic ladders are a prerequisite for reliable genotyping of short tandem repeat (STR) polymorphisms and consistent results across instrument platforms. For eight STR-loci located on the short arm of chromosome 6 (6p21.3), a sequenced based nomenclature was established according to international recommendations. Publicly available reference DNA samples were sequenced enabling interested laboratories to construct their own allelic ladders. Three tetrameric (D6S2691, D6S2678, DQIV), one trimeric (D6S2906) and four dimeric repeat loci (D6S2972, D6S2792, D6S2789, D6S273) were investigated. Apart from the very complex sequence structure at the DQIV locus, three loci showed a compound and four loci a simple repeat pattern. In the flanking regions of some loci additional single nucleotide and insertion/deletion polymorphisms occurred as well as sequence polymorphisms within the repeat region of alleles with the same length. In an Austrian Caucasoid population sample (n=293) between eight and 22 alleles were found. No significant deviation from Hardy-Weinberg expectations was observed, the power of discrimination ranged from 0.826 to 0.978. The loci cover the HLA-coding region from HLA-A to HLA-DQB1 and can be used for a better definition of HLA haplotypes for population and disease association studies, recombination point mapping, haematopoietic stem cell transplantation as well as for identity and relationship testing.

  2. Interaction Between Hippocampus and Cerebellum Crus I in Sequence-Based but not Place-Based Navigation.

    PubMed

    Iglói, Kinga; Doeller, Christian F; Paradis, Anne-Lise; Benchenane, Karim; Berthoz, Alain; Burgess, Neil; Rondi-Reig, Laure

    2015-11-01

    To examine the cerebellar contribution to human spatial navigation we used functional magnetic resonance imaging and virtual reality. Our findings show that the sensory-motor requirements of navigation induce activity in cerebellar lobules and cortical areas known to be involved in the motor loop and vestibular processing. By contrast, cognitive aspects of navigation mainly induce activity in a different cerebellar lobule (VIIA Crus I). Our results demonstrate a functional link between cerebellum and hippocampus in humans and identify specific functional circuits linking lobule VIIA Crus I of the cerebellum to medial parietal, medial prefrontal, and hippocampal cortices in nonmotor aspects of navigation. They further suggest that Crus I belongs to 2 nonmotor loops, involved in different strategies: place-based navigation is supported by coherent activity between left cerebellar lobule VIIA Crus I and medial parietal cortex along with right hippocampus activity, while sequence-based navigation is supported by coherent activity between right lobule VIIA Crus I, medial prefrontal cortex, and left hippocampus. These results highlight the prominent role of the human cerebellum in both motor and cognitive aspects of navigation, and specify the cortico-cerebellar circuits by which it acts depending on the requirements of the task.

  3. Novel Sequence-Based Mapping of Recently Emerging H5NX Influenza Viruses Reveals Pandemic Vaccine Candidates

    PubMed Central

    Anderson, Christopher S.; DeDiego, Marta L.; Thakar, Juilee; Topham, David J.

    2016-01-01

    Recently, an avian influenza virus, H5NX subclade 2.3.4.4, emerged and spread to North America. This subclade has frequently reassorted, leading to multiple novel viruses capable of human infection. Four cases of human infections, three leading to death, have already occurred. Existing vaccine strains do not protect against these new viruses, raising a need to identify new vaccine candidate strains. We have developed a novel sequence-based mapping (SBM) tool capable of visualizing complex protein sequence data sets using a single intuitive map. We applied SBM on the complete set of avian H5 viruses in order to better understand hemagglutinin protein variance amongst H5 viruses and identify any patterns associated with this variation. The analysis successfully identified the original reassortments that lead to the emergence of this new subclade of H5 viruses, as well as their known unusual ability to re-assort among neuraminidase subtypes. In addition, our analysis revealed distinct clusters of 2.3.4.4 variants that would not be covered by existing strains in the H5 vaccine stockpile. The results suggest that our method may be useful for pandemic candidate vaccine virus selection. PMID:27494186

  4. Measurement of atmospheric radicals by chemical amplification

    NASA Astrophysics Data System (ADS)

    Arias, Maria Corina

    1998-11-01

    In this work, atmospheric radicals were measured using the chemical amplification technique. To calibrate the chemical amplifier, an UV water photolysis radical source was built and tested. This source proved to be reliable and portable, and capable of delivering radical concentrations within the range of values found in the troposphere. We tested the performance of our instrument at the Peroxy Radical InterComparison Exercise II (PRICE II). In this intercomparison seven chemical amplifiers participated measuring several HO2 and CH3O2 concentrations. Results from this campaign indicate that all of the chemical amplifiers are equally capable of measuring HO2 and CH3O2 radicals from two different radical sources (ICG-HO2 source and UEA- CH3O2 source). The average response towards the ICG and UEA sources were 70% and 45%, respectively. Losses in the delivery system are thought to be responsible for these low responses. Radical measurements were taken at 4 contrasting sites: Atlantic '96 (clean continental), SONTOS '92 and '93 (rural), Calabozo '93 (tropical clean continental), and Pacific '93 (predominantly urban), where maximum ROx concentrations ranged from 17 to 52 pptv. These values are consistent with those found in the literature for similar regions. The measured radical concentrations reflect the interaction between the main production and loss processes at the different sites, as for example ozone photolysis and HNO3 formation. At Calabozo, the combination of moderate O3, low NOx and small Zenith angles resulted in the highest ROx measured. At the Pacific '93 site, O3 is higher, but NOx concentrations are also very high, enhancing the radical loss processes, and explaining the moderate radical concentrations observed. At Atlantic '96 the very low NOx concentration might account for the radical concentrations observed, even in the presence of low O3 concentrations. At SONTOS, the highest ozone concentrations were observed, so we would expect the radical

  5. Regulation of ribosomal DNA amplification by the TOR pathway

    PubMed Central

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

    2015-01-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions. PMID:26195783

  6. Active beam shaping in multi-levels amplification system

    NASA Astrophysics Data System (ADS)

    Zhao, Tianzhuo; Fan, Zhongwei; Qiu, Jisi; Tang, Xiongxin; Lin, Weiran; Zhang, Hongbo

    2016-09-01

    Using Liquid Crystal Spatial Light Modulator (LC-SLM) as a beam shaping device to improve beam quality in high-gain amplification system is reported. 1.6 nJ injected small-size signal Gaussian beam can be amplified to 5 J by 4 stages amplification, and finally output beam is a 50mm×50mm square spot with flat-top intensity distribution. In the amplification system we designed, LC-SLM is placed after the second level of amplifier, where the signal laser energy is about 20mJ, and beam size is 10mm×10mm. The structure of Fourier image transfer is also implemented in this amplifications system to be capable of maintaining high-quality image transmission in the amplification process. The LC-SLM as an object, is imaged by beam expand lenses and spatial filters lenses in the amplifications system to get good quality of imaging. By catching output spot and making a feed-back, transmission efficiency of each pixel on LC-SLM is modulated, high energy density area can be decreased to realize flat-top intensity distribution. A spot modulation function is defined as, using the maximum grey value on spot area divided by the average grey value of the image after background correction. By this, amplified laser obtains the spot modulation of 1.24 on central 90% area of the spot. Furthermore, un-uniform distribution on the full spot, soften effects of spot edge, and output beam shape can also be optimized by the LC-SLM shaping scheme in the amplification system.

  7. Alternative Chemical Amplification Methods for Peroxy Radical Detection

    NASA Astrophysics Data System (ADS)

    Wood, E. C. D.

    2014-12-01

    Peroxy radicals (HO2, CH3O2, etc.) are commonly detected by the chemical amplification technique, in which ambient air is mixed with high concentrations of CO and NO, initiating a chain reaction that produces 30 - 200 NO2 molecules per sampled peroxy radical. The NO2 is then measured by one of several techniques. With the exception of CIMS-based techniques, the chemical amplification method has undergone only incremental improvements since it was first introduced in 1982. The disadvantages of the technique include the need to use high concentrations of CO and the greatly reduced sensitivity of the amplification chain length in the presence of water vapor. We present a new chemical amplification scheme in which either ethane or acetaldehyde is used in place of CO, with the NO2 product detected using Cavity Attenuated Phase Shift spectroscopy (CAPS). Under dry conditions, the amplification factor of the alternative amplifiers are approximately six times lower than the CO-based amplifier. The relative humidity "penalty" is not as severe, however, such that at typical ambient relative humidity (RH) values the amplification factor is within a factor of three of the CO-based amplifier. Combined with the NO2 sensitivity of CAPS and a dual-channel design, the detection limit of the ethane amplifier is less than 2 ppt (1 minute average, signal-to-noise ratio 2). The advantages of these alternative chemical amplification schemes are improved safety, a reduced RH correction, and increased sensitivity to organic peroxy radicals relative to HO2.

  8. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic...

  9. 21 CFR 866.3225 - Enterovirus nucleic acid assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Enterovirus nucleic acid assay. 866.3225 Section... nucleic acid assay. (a) Identification. An enterovirus nucleic acid assay is a device that consists of primers, probes, enzymes, and controls for the amplification and detection of enterovirus ribonucleic...

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

    PubMed

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

    2000-10-01

    The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.

  11. Optical Parametric Amplification for High Peak and Average Power

    SciTech Connect

    Jovanovic, Igor

    2001-11-26

    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

  12. Local seismic site amplification: effects of obliquely incident antiplane motions

    NASA Astrophysics Data System (ADS)

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

    2016-11-01

    Seismic site amplification studies are generally used to assess the effects of local geology and soil conditions on ground motion characteristics. Although extensive reviews on site amplification phenomena associated with stratigraphic effects can be found in the specialized literature, it should be pointed out that most of the practical applications have been limited to the study of vertically propagating shear horizontal (SH) waves, i.e., to the 1-D soil amplification problem. Furthermore, little attention, if any, has been devoted to the study of the effects of non-vertically incident SH waves on surface accelerograms and on the earthquake response of structures. In the present work, the study is extended to an investigation of 2-D site amplification of non-vertically propagating seismic shear waves in multilayered viscoelastic soil deposits. Sensitivity analyses of the effects of non-vertical incidence on site amplification functions are performed based on site geotechnical data collected from post-seismic investigations of the 1980 El-Asnam earthquake. Analytical results are discussed in terms of seismic site transfer functions, spectral ratios, surface acceleration time histories, and structural response spectra for different values of wave incidence angle. Both bedrock and rock outcropping cases are examined.

  13. Loopback rolling circle amplification for ultrasensitive detection of Kras gene.

    PubMed

    Xu, Huo; Wu, Dong; Jiang, Yifan; Zhang, Rongbo; Wu, Qingzheng; Liu, Yiyun; Li, Feng; Wu, Zai-Sheng

    2017-03-01

    Mutations in Kras gene may be used as a diagnostic marker and a target for treatment of the broad spectrum of human cancers. In this study, we developed a new class of amplification assay, double-hairpin molecular beacon (DHMB)-based cascade rolling circle amplification (RCA), for ultrasensitive and selective detection of Kras gene in a homogenous solution. Specifically, target DNA can hybridize with DHMB and activate cyclical target strand-displacement polymerization (CTDP) and nicking-mediated strand-displacement polymerization (NMDP). The resulting nicked/displaced fragments substantially outnumber target DNA and cause the cascade rolling circle amplification (C-RCA) and nicked fragment-induced strand-displacement polymerization (NFDP). Even if four amplification processes are designed, only DHMB, padlock probe and polymerization primer are involved. Under optimized conditions, this screening system exhibits a linear range of 5 orders of magnitude (from 100fM to 20nM), and the detection limit is down to 16fM. Moreover, the developed biosensing system offers a high assay specificity for perfectly matched target DNA, and the measured data from practical samples demonstrated the potential application in the cancer diagnoses. As a proof-of-concept genetic assay, the novel signaling strategy, as well as desirable analytical capability, would significantly benefit the development of versatile amplification gene profiling platforms, revealing great promise in biological studies and medical diagnostics.

  14. Optimizing direct amplification of forensic commercial kits for STR determination.

    PubMed

    Caputo, M; Bobillo, M C; Sala, A; Corach, D

    2017-04-01

    Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman(®) 3 MM paper, FTA™ Classic cards, and Whatman(®) Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective.

  15. Problems encountered when defining Arctic amplification as a ratio

    PubMed Central

    Hind, Alistair; Zhang, Qiong; Brattström, Gudrun

    2016-01-01

    In climate change science the term ‘Arctic amplification’ has become synonymous with an estimation of the ratio of a change in Arctic temperatures compared with a broader reference change under the same period, usually in global temperatures. Here, it is shown that this definition of Arctic amplification comes with a suite of difficulties related to the statistical properties of the ratio estimator itself. Most problematic is the complexity of categorizing uncertainty in Arctic amplification when the global, or reference, change in temperature is close to 0 over a period of interest, in which case it may be impossible to set bounds on this uncertainty. An important conceptual distinction is made between the ‘Ratio of Means’ and ‘Mean Ratio’ approaches to defining a ratio estimate of Arctic amplification, as they do not only possess different uncertainty properties regarding the amplification factor, but are also demonstrated to ask different scientific questions. Uncertainty in the estimated range of the Arctic amplification factor using the latest global climate models and climate forcing scenarios is expanded upon and shown to be greater than previously demonstrated for future climate projections, particularly using forcing scenarios with lower concentrations of greenhouse gases. PMID:27461918

  16. Role of Surface Temperature Inversions in Arctic Amplification

    NASA Astrophysics Data System (ADS)

    Lesins, G. B.; Duck, T. J.; Drummond, J. R.

    2010-12-01

    Arctic surface temperatures are warming at a rate several times larger than the global average in a phenomenon called Arctic Amplification. Although a number of feedback mechanisms, including sea ice reduction, enhanced meridional heat transport and changing aerosol and cloud properties, have been proposed to explain the amplification, the role of low level atmospheric stability has not received as much attention. The Arctic atmospheric boundary layer typically possesses a surface-based temperature inversion that is particularly stable and deep during the winter half of the year. The presence of the inversion stratifies the boundary layer inhibiting the vertical transport of sensible and latent heats. Hence the surface temperature response to a perturbation in the downward radiative forcing is enhanced by the presence of a stable surface layer. By using the operational surface weather observations and tropospheric soundings it was found that the magnitude of the surface temperature warming from 1984 to 2007 at Eureka (80N, 86.2W), in the Canadian High Arctic, has a large seasonal dependence that is linked to the strength of the surface-based temperature inversion. The Arctic Amplification Factor at Eureka in this time period is ~5x. The enhanced warming in the presence of a stronger inversion is an important contributor to the Arctic amplification phenomenon. The inversion augments the amplification caused by forcing and feedback mechanisms associated with regional climate change. Partitioning the data set according to cloud cover and surface wind speeds helps to demonstrate more clearly the role of the inversion.

  17. Clinical application of multiple displacement amplification in preimplantation genetic diagnosis.

    PubMed

    Hellani, Ali; Coskun, Serdar; Tbakhi, Abdelghhani; Al-Hassan, Saad

    2005-03-01

    Multiple displacement amplification (MDA) is a technique used in the amplification of very small amounts of DNA. MDA is reported to yield large quantities of high-quality DNA. The applicability of MDA to single cells was recently demonstrated as a potential technique for preimplantation genetic diagnosis (PGD). This paper shows the first clinical application of MDA in PGD. Two cycles of PGD were performed in two diseases, resulting in two pregnancies. All the diagnoses given on blastomeres were confirmed on the non-transferred whole embryos. The blastomere diagnosis was coupled with short tandem repeat (STR) analysis (16 loci) in all cycles. Allelic drop-out (ADO) assessment and amplification efficiency were evaluated on 40 single lymphocytes derived from parents of each disease. ADO and amplification failure were 10.3 and 2.2% for beta-thalassaemia and 17.9 and 2.2% for cystic fibrosis respectively. HLA matching for A, B and DR was performed successfully on single cell for the beta-thalassaemia family using similar methods to genomic DNA. The PGD protocol used in all diseases con