title: Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using Salmonella as a model organism.
abstract: 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.
authors.affiliation: Division of Microbiology, GBF-German Research Center for Biotechnology, Braunschweig, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Fey
authors.forename: Stefan
authors.initials: S
authors.lastname: Eichler
authors.forename: Sébastien
authors.initials: S
authors.lastname: Flavier
authors.forename: Richard
authors.initials: R
authors.lastname: Christen
authors.forename: Manfred G
authors.initials: MG
authors.lastname: Höfle
authors.forename: Carlos A
authors.initials: CA
authors.lastname: Guzmán
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015169:Colony Count, Microbial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000592:standards; D004784:Environmental Monitoring / Q000379:methods; D005618:Fresh Water / Q000382:microbiology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012329:RNA, Bacterial / Q000235:genetics* / Q000592:standards*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012486:Salmonella typhimurium / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C075518:invA protein, Bacteria