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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.



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