20 research outputs found
Eprobes in multiplex detection.
<p>Regions of the human EGFR and KRAS genes were amplified from genomic DNA and amplification was monitored by specific Eprobes having different dyes. A: Amplification curves plotting random fluorescent units (RFU) obtained from LightCycler 480 against PCR cycle number (Red: genomic DNA, Green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature (Red: genomic DNA, Green: negative control). Plots in yellow on the left show signals for KRAS using dye D514 (Eprobe KWT14m3 TO); plots in pink on the right show signals for EGFR using dye D570 (Eprobe 215-21 wt TP).</p
Eprobe derived background.
<p>A. Eprobe 215-21 wt TO labeled with D514 was digested as described in the Materials and Methods and fluorescence excitation spectra from 495 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). B. Eprobe 215-21 wt TP labeled with dye D570 was digested as described in the Materials and Methods and fluorescence excitation spectra from 550 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). C. Plotting random fluorescent units (RFU, mean values from triplicate data for each experiment) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against plasmid DNA template concentration (from 150 to 150,000,000 copies per reaction). Eprobe concentrations are indicated by the different colors (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). D. Plotting random fluorescent units (RFU) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against Eprobe concentration (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). For each data point, mean values plus error bars from triplicate values for each experiment and for all template concentrations are given in the graph.</p
Agarose gel electrophoresis of PCR products.
<p>A: PCR products from real-time PCR experiments were analyzed on a 3% agarose gel. Lanes: 1: TaqMan Probe, 2:, Eprobe 203-10 wt TO 3: C3-blocked 203-10 wt oligonucleotide, 4: Eprobe 205-13 wt TO, 5: Eprobe205-13m TO, 6: C3-blocked 205-13 wt oligonucleotide, 7: Eprobe 215-21 wt TO, 8: Eprobe 215-21m TO, 9: C3-blocked 215-21 wt oligonucleotide, 10: Eprobe 215-21 wt TP, 11: SYBR Green I, M: marker. B: Partial sequence of human EGFR gene. Location of the primers (blue boxes) and the mutation L858R (red box) are indicated.</p
Eprobes used for this study.
<p>(T<sub>M</sub> values for TO-labeled Eprobes were estimated using the following settings: 0.05 M Na<sup>+</sup>, 0.002 M Mg<sup>2+</sup>, 0.2 µM Eprobe, 60°C Temperature; *T<sub>M</sub> values for Eprobe 215-21 wt TP are actual values; in Eprobe name: TO = D514, TP = D570; Z indicating position of modified T).</p
Eprobe mediated mutation detection.
<p>Asymmetric PCR was performed to enrich the reverse strand for mutation detection using different templates and Eprobes. For further details refer to Materials and Methods. A: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 5×10<sup>4</sup> copies of plasmid DNA using Eprobe 205-13 wt TO. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 1 ng genomic DNA using Eprobe 215-21 wt TP. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control).</p
Structure and function of Eprimer and Eprobe.
<p>A. Chemical structure of modified deoxythymidine carrying two dye moieties. B. Chemical structure of D514 “Thiazole Orange”. C. Chemical structure of D570 “Thiazole Pink”. D. Signal generation by an arbitrary Eprimer (ECHO), where the two dye moieties are paired in the single-stranded oligonucleotide for signal suppression. Upon hybridization to complementary strand, dye moieties get separated and intercalate into the double-strand leading to emission of strong fluorescence. E. Structure of an arbitrary Eprobe having a blocked 3′ end to prevent primer extension during PCR.</p
Distribution of antimicrobial resistance genes among ESBL-producing <i>Enterobacter</i> spp.
*<p>All <i>qnrA</i> were <i>qnrA1</i>.</p
Dendrogram and PFGE of <i>Xba</i>I-digested genomic DNAs from ESBL-producing <i>Enterobacter</i> species.
<p>EC: <i>Enterobacter cloacae</i>, EA: <i>Enterobacter aerogenes</i>. Strain No. and PFGE type correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037967#pone-0037967-t002" target="_blank">Table 2</a>.</p
Profile of <i>Enterobacter</i> spp. carrying ESBL genes and results of the boronic acid disk test.
<p>CTX: cefotaxime, CAZ: ceftazidime, FOX: cefoxitin, FEP: cefepime, MEM: meropenem, LVX: levofloxacin, AMK: amikacin, BA: 3-aminophenylboronic acid. EC: <i>Enterobacter cloacae</i>, EA: <i>Enterobacter aerogenes.</i></p
MLST and PFGE of <i>Xba</i>I-digested genomic DNA from CTX-M-15- and CTX-M-27-producing <i>E. coli</i> strains.
<p>The 9 CTX-M-15-producing isolates were divided into 8 unique PFGE types. The 21 CTX-M-27-producing isolates were divided into 14 unique PFGE types, with the most common type being found in 6 patients. M1∼4: Lambda Ladder.</p