scholarly journals Comparison of sandwich hybridization assay and quantitative PCR for the quantification of live and preserved cultures ofHeterosigma akashiwo(Raphidophyceae)

2014 ◽  
Vol 12 (4) ◽  
pp. 232-245 ◽  
Author(s):  
Cameron Doll ◽  
Christopher R. Main ◽  
Colleen Bianco ◽  
Kathryn J. Coyne ◽  
Dianne I. Greenfield
Keyword(s):  
Quantitative Pcr ◽  
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

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.

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.

Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay

2005 ◽  
Vol 62 (2) ◽  
pp. 167-179 ◽  
Author(s):  
Tarja Leskelä ◽  
Anu Tilsala-Timisjärvi ◽  
Jaana Kusnetsov ◽  
Peter Neubauer ◽  
Antje Breitenstein
Keyword(s):  
16S Rrna ◽  
Hybridization Assay ◽  
Specific Detection ◽  
Sandwich Hybridization ◽  
Sandwich Hybridization Assay
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