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.

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic

“…The thermal cycling program was performed as follows: an initial denaturation step at 95 1C for 3 min, followed by 45 cycles at 95 1C for 10 s and 60 1C for 30 s. The PCR products were analyzed on a 2.0% agarose gel, stained with ethidium bromide and visualized under ultraviolet light. Copy numbers of 16E5 cDNA were determined as described (Fey et al, 2004). Real-time quantitation was performed with the help of the iCycler IQ optical system software version 3.0a (Bio-Rad), according to the manufacturer's manual.…”

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

“…Norovirus particles can only be obtained from infected individuals making it very difficult to obtain viral particles for use as standards. A more important advantage is that it provides an RNA standard for all targeted RNA viruses in one reagent, as having an RNA standard is essential for accurate quantification of RNA 36 . However, its ability to quantify virus accurately is limited by the fact that matrix effects are not taken into account.…”

Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

“…DNA sequence), subsequently failing to ensure proper specificity of PCR product(s) (Rahn et al, 1992;. Additionally, we employed TaqMan probes for our qPCR assay which had several advantages over the use of nonspecific (although cheaper) SYBR Green I assays, including greater sensitivity and a probe-based sequence-specific verification of PCR product identity (Wittwer et al, 1997;Fey et al, 2004;Jacobsen and Holben, 2007). …”