This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fey, A.
Right arrow Articles by Guzmán, C. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fey, A.
Right arrow Articles by Guzmán, C. A.
Agricola
Right arrow Articles by Fey, A.
Right arrow Articles by Guzmán, C. A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, June 2004, p. 3618-3623, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3618-3623.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Establishment of a Real-Time PCR-Based Approach for Accurate Quantification of Bacterial RNA Targets in Water, Using Salmonella as a Model Organism

Axel Fey,1 Stefan Eichler,1 Sébastien Flavier,2 Richard Christen,2 Manfred G. Höfle,1 and Carlos A. Guzmán1*

Division of Microbiology, GBF-German Research Center for Biotechnology, Braunschweig, Germany,1 UMR 6543 CNRS and Université Nice Sophia Antipolis Centre de Biochimie, F-06108 Nice, France2

Received 17 December 2003/ Accepted 3 March 2004

Quantitative PCR (Q-PCR) is a fast and efficient tool to quantify target genes. In eukaryotic cells, quantitative reverse transcription-PCR (Q-RT-PCR) is also used to quantify gene expression, with stably expressed housekeeping genes as standards. In bacteria, such stable expression of housekeeping genes does not occur, and the use of DNA standards leads to a broad underestimation. Therefore, an accurate quantification of RNA is feasible only by using appropriate RNA standards. We established and validated a Q-PCR method which enables the quantification of not only the number of copies of target genes (i.e., the number of bacterial cells) but also the number of RNA copies. The genes coding for InvA and the 16S rRNA of Salmonella enterica serovar Typhimurium were selected for the evaluation of the method. As DNA standards, amplified fragments of the target genes were used, whereas the same DNA standards were transcribed in vitro for the development of appropriate RNA standards. Salmonella cultures and environmental water samples inoculated with bacteria were then employed for the final testing. Both experimental approaches led to a sensitive, accurate, and reproducible quantification of the selected target genes and RNA molecules by Q-PCR and Q-RT-PCR. It is the first time that RNA standards have been successfully used for a precise quantification of the number of RNA molecules in prokaryotes. This demonstrates the potential of this approach for determining the presence and metabolic activity of pathogenic bacteria in environmental samples.

* Corresponding author. Mailing address: Division of Microbiology, GBF-German Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Phone: 49-531-6181558. Fax: 49-531-6181411. E-mail: cag{at}gbf.de.

Applied and Environmental Microbiology, June 2004, p. 3618-3623, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3618-3623.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Rodriguez-Martinez, J.-M., Poirel, L., Nordmann, P. (2009). Molecular Epidemiology and Mechanisms of Carbapenem Resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 53: 4783-4788 [Abstract] [Full Text]  
  • Fallschissel, K., Kampfer, P., Jackel, U. (2009). Direct Detection of Salmonella Cells in the Air of Livestock Stables by Real-Time PCR. ANN OCCUP HYG 53: 859-868 [Abstract] [Full Text]  
  • Welander, P. V., Hunter, R. C., Zhang, L., Sessions, A. L., Summons, R. E., Newman, D. K. (2009). Hopanoids Play a Role in Membrane Integrity and pH Homeostasis in Rhodopseudomonas palustris TIE-1. J. Bacteriol. 191: 6145-6156 [Abstract] [Full Text]  
  • Gonzalez-Escalona, N., Hammack, T. S., Russell, M., Jacobson, A. P., De Jesus, A. J., Brown, E. W., Lampel, K. A. (2009). Detection of Live Salmonella sp. Cells in Produce by a TaqMan-Based Quantitative Reverse Transcriptase Real-Time PCR Targeting invA mRNA. Appl. Environ. Microbiol. 75: 3714-3720 [Abstract] [Full Text]  
  • Girlich, D., Poirel, L., Nordmann, P. (2009). CTX-M Expression and Selection of Ertapenem Resistance in Klebsiella pneumoniae and Escherichia coli. Antimicrob. Agents Chemother. 53: 832-834 [Abstract] [Full Text]  
  • Perez-Osorio, A. C., Franklin, M. J. (2008). qRT-PCR of Microbial Biofilms. CSH Protocols 2008: pdb.prot5066-pdb.prot5066 [Abstract] [Full Text]  
  • Bishop, A., House, D., Perkins, T., Baker, S., Kingsley, R. A., Dougan, G. (2008). Interaction of Salmonella enterica serovar Typhi with cultured epithelial cells: roles of surface structures in adhesion and invasion. Microbiology 154: 1914-1926 [Abstract] [Full Text]  
  • Dunkley, K. D., McReynolds, J. L., Hume, M. E., Dunkley, C. S., Callaway, T. R., Kubena, L. F., Nisbet, D. J., Ricke, S. C. (2007). Molting in Salmonella Enteritidis-Challenged Laying Hens Fed Alfalfa Crumbles. I. Salmonella Enteritidis Colonization and Virulence Gene hilA Response. Poult. Sci. 86: 1633-1639 [Abstract] [Full Text]  
  • Smith, C. J., Nedwell, D. B., Dong, L. F., Osborn, A. M. (2007). Diversity and Abundance of Nitrate Reductase Genes (narG and napA), Nitrite Reductase Genes (nirS and nrfA), and Their Transcripts in Estuarine Sediments. Appl. Environ. Microbiol. 73: 3612-3622 [Abstract] [Full Text]  
  • Cheng, P., Nefedova, Y., Corzo, C. A., Gabrilovich, D. I. (2007). Regulation of dendritic-cell differentiation by bone marrow stroma via different Notch ligands. Blood 109: 507-515 [Abstract] [Full Text]  
  • Nocker, A., Camper, A. K. (2006). Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide.. Appl. Environ. Microbiol. 72: 1997-2004 [Abstract] [Full Text]  
  • Johnson, D. R., Lee, P. K. H., Holmes, V. F., Alvarez-Cohen, L. (2005). An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the tceA Reductive Dehalogenase Gene. Appl. Environ. Microbiol. 71: 3866-3871 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»