Colorimetric Detection of Anthrax DNA with a Peptide Nucleic Acid Sandwich-Hybridization Assay

2007 ◽  
Vol 129 (27) ◽  
pp. 8424-8425 ◽  
Author(s):  
Ning Zhang ◽  
Daniel H. Appella
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Colorimetric Detection ◽  
Hybridization Assay ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay

A novel high throughput nucleic acid sandwich hybridization assay on a gold substrate

2013 ◽  
Vol 5 (11) ◽  
pp. 2835 ◽  
Author(s):  
C. H. van den Kieboom ◽  
T. S. Y. van Domburg ◽  
M. I. de Jonge ◽  
G. Ferwerda ◽  
P. W. M. Hermans
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
Hybridization Assay ◽  
Gold Substrate ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay

High Affinity γPNA Sandwich Hybridization Assay for Rapid Detection of Short Nucleic Acid Targets with Single Mismatch Discrimination

2013 ◽  
Vol 14 (7) ◽  
pp. 2253-2261 ◽  
Author(s):  
Johnathan M. Goldman ◽  
Li Ang Zhang ◽  
Arunava Manna ◽  
Bruce A. Armitage ◽  
Danith H. Ly ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Rapid Detection ◽  
Hybridization Assay ◽  
High Affinity ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay ◽  
Mismatch Discrimination ◽  
Single Mismatch

scholarly journals Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

2008 ◽  
Vol 74 (24) ◽  
pp. 7463-7470 ◽  
Author(s):  
Daniel Thieme ◽  
Peter Neubauer ◽  
Dietrich H. Nies ◽  
Gregor Grass
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Time Course ◽  
Copper Ions ◽  
Copper Resistance ◽  
Hybridization Assay ◽  
Target Mrna ◽  
Cell Extracts ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay

ABSTRACT Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.

Hybridization assay of hepatitis B virus by QCM peptide nucleic acid biosensor

2008 ◽  
Vol 23 (6) ◽  
pp. 879-885 ◽  
Author(s):  
Chunyan Yao ◽  
Tangyou Zhu ◽  
Jin Tang ◽  
Rong Wu ◽  
Qinghai Chen ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Nucleic Acid ◽  
Hepatitis B ◽  
Peptide Nucleic Acid ◽  
Hybridization Assay ◽  
B Virus

scholarly journals Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

2008 ◽  
Vol 74 (23) ◽  
pp. 7297-7305 ◽  
Author(s):  
Xu Li ◽  
Eberhard Morgenroth ◽  
Lutgarde Raskin
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Peptide Nucleic Acid ◽  
Molecular Beacon ◽  
Complex Mixture ◽  
Hybridization Assay ◽  
Pure Cultures ◽  
Quantitative Results ◽  
The Difference ◽  
Different Levels

ABSTRACT The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

Detection of microcystin-producingMicrocystisin Guanqiao Lake using a sandwich hybridization assay

2012 ◽  
Vol 58 (4) ◽  
pp. 442-447 ◽  
Author(s):  
Jing Ping Zhu ◽  
Shi Du ◽  
Xian Li
Keyword(s):  
Calibration Curve ◽  
Cell Number ◽  
Hybridization Assay ◽  
Sequence Analyses ◽  
Sandwich Hybridization ◽  
Cyanobacterial Culture ◽  
Field Samples ◽  
Laboratory Cultures ◽  
Pcr Technology ◽  
Sandwich Hybridization Assay

Based on sequence analyses of the mcyJ gene from Microcystis strains, a probe pair TJF and TJR was designed and a sandwich hybridization assay (SHA) was established to quantitatively detect microcystin-producing Microcystis. Through BLAST and cyanobacterial culture tests, TJF and TJR were demonstrated to be specific for microcystin-producing Microcystis. A calibration curve for the SHA was established, and the lowest detected concentration was 100 cells·mL–1. Laboratory cultures and field samples from Guanqiao Lake were analyzed with both the SHA and microscopy. The cell number of microcystin-producing Microcystis and that of total Microcystis were compared. The biotic and abiotic components of the samples were of little disturbance to the SHA. In this study, a SHA was established to detect Microcystis, providing an alternative to PCR–ELISA and real-time PCR technology.

scholarly journals Peptide Nucleic Acid–Based In Situ Hybridization Assay for Detection of Parvovirus B19 Nucleic Acids

2006 ◽  
Vol 52 (6) ◽  
pp. 973-978 ◽  
Author(s):  
Francesca Bonvicini ◽  
Claudia Filippone ◽  
Elisabetta Manaresi ◽  
Giovanna Angela Gentilomi ◽  
Marialuisa Zerbini ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Nucleic Acids ◽  
Peptide Nucleic Acid ◽  
Parvovirus B19 ◽  
Hybridization Assay ◽  
Pcr Assay ◽  
Clinical Specimens ◽  
Rna Molecules

Abstract Background: Peptide nucleic acid (PNA) molecules are known to bind complementary nucleic acid sequences with a much stronger affinity and with more stable binding than DNA or RNA molecules. We chose parvovirus B19, which is diagnosed by detection of nucleic acids by in situ hybridization assay (ISH) and/or PCR, as an experimental model to develop an ISH assay that uses biotinylated PNA probes to detect viral genome in clinical specimens. Methods: We first optimized the PNA-ISH assay on B19-infected and mock-infected UT-7/EpoS1 cells and then tested the assay on archival B19 specimens and on consecutive specimens. All data were compared with data obtained with a standardized DNA-based ISH assay and confirmed by a PCR-ELISA. Results: PNA-ISH detected B19 genome in a higher number of B19-infected UT-7/EpoS1 cells and with a more defined localization of viral nucleic acids than the standardized DNA-ISH assay. Moreover, PNA-ISH was able to detect B19 genome in all positive archival samples, whereas DNA-ISH failed in 5 samples. PNA-ISH detected more positive samples than DNA-ISH when consecutive specimens were analyzed, and a close agreement was found with PCR-ELISA results. Conclusions: The PNA-ISH assay had sensitivity and specificity comparable to a PCR assay and was more practical and quicker to perform than standard hybridization assays. The assay may be a suitable diagnostic test for the detection of viral nucleic acids in clinical specimens.

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