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.

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 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.

Impact of a rapid peptide nucleic acid fluorescence in situ hybridization assay on treatment of Candida infections

2012 ◽  
Vol 69 (21) ◽  
pp. 1910-1914 ◽  
Author(s):  
Emily L. Heil ◽  
Lindsay M. Daniels ◽  
Dustin M. Long ◽  
Kyle G. Rodino ◽  
David J. Weber ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Fluorescence In Situ Hybridization ◽  
Peptide Nucleic Acid ◽  
Hybridization Assay ◽  
Candida Infections

scholarly journals Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

2003 ◽  
Vol 69 (9) ◽  
pp. 5673-5678 ◽  
Author(s):  
Chuanwu Xi ◽  
Michal Balberg ◽  
Stephen A. Boppart ◽  
Lutgarde Raskin
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Molecular Beacon ◽  
Signal To Noise Ratio ◽  
Microfluidic Devices ◽  
Molecular Beacons ◽  
Target Cells ◽  
Signal To Noise ◽  
Whole Cells

ABSTRACT DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution. In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB. DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step. The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons. Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data. Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices.

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