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Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays
Citation:
Pitkanen, T., H. Ryu, M. Elk, A. Hokajarvi, S. Siponen, A. Vepsalainen, P. Rasanen, AND J. Santodomingo. Detection of fecal bacteria and source tracking identifiers in environmental waters using rRNA-based RT-qPCR and rDNA-based qPCR assays. ENVIRONMENTAL SCIENCE & TECHNOLOGY. American Chemical Society, Washington, DC, 47(23):13611-13620, (2013).
Impact/Purpose:
The objectives of this study were to further compare the detection frequency of the different rRNA gene-targeting assays using both RNA and DNA as templates, and to evaluate the distribution of targeted fecal bacteria, including source-specific markers, across an urban watershed impacted by sewage pollution. Additionally, we studied the correlation between human-specific Bacteroidales markers and conventional fecal indicators using rRNA based RT-qPCR to better evaluate the efficacy of using these markers for the detection of human fecal pollution.
Description:
The identification of fecal pollution sources is commonly performed using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as “naked DNA” in the environment. To this end, we compared the detection frequency of host specific markers and fecal bacteria using RNA and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. The detection of the targeted bacteria was higher in most sites using RNA-based methods. Specifically, 8%, 87%, and 85% of the samples positive for general enterococci, E. faecalis, and E. faecium markers, respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. One average, two human-specific Bacteriodales markers were not detected when using DNA in 12% of the samples while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays that in qPCR assays. In general, the results from this study suggest that RNA-based assays can increase the detection of fecal bacteria in water samples impacted with fecal sources.
