Detection of Fecal Bacteria and Source Tracking Identifiers in Environmental Waters Using rRNA-Based RT-qPCR and rDNA-Based qPCR Assays

Abstract

In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.

Distribution of Human-Specific Bacteroidales and Fecal Indicator Bacteria in an Urban Watershed Impacted by Sewage Pollution, Determined Using RNA- and DNA-Based Quantitative PCR Assays

“…The linear range of quantification for the qPCR assay of human-specific Bacteroidales (HF183 and BacHum) and E. coli markers were between 10 and 10 5 copies, while the linear range for qPCR assay of Entero1 was 50 to 5 ϫ 10 5 copies, and those for Faecalis and Faecium1 were between 10 2 and 10 5 copies. PCR inhibition tests were performed with 10-fold dilutions of each DNA extract as described in Pitkänen et al (27). In these tests, a C T value proportional to a 10-fold dilution relative to the undiluted DNA templates resulted, suggesting that PCR inhibition did not interfere with the amplification efficiency.…”

“…RNA and DNA were extracted from the membranes as described elsewhere (27). Briefly, frozen membranes were subjected to bead-beating using DNase and RNase free glass beads (Mo Bio Laboratories, Inc., Carlsbad, CA) in the presence of lysis buffer (Buffer RLT Plus; Qiagen GmbH, Hilden, Germany) containing ␤-mercaptoethanol (Sigma-Aldrich Co., St. Louis, MO).…”

“…The concentration and purity of RNA and DNA was determined by using Qubit RNA and dsDNA HS assay kits and a Qubit 2.0 Fluorometer (Life Technologies). cDNA was synthesized on the extraction day from the purified RNA extracts using random hexamer primed Superscript III system for RT-PCR as described previously (27). Both cDNA and DNA were stored at Ϫ20°C for subsequent analyses.…”

“…The range of quantification and the limit of detection for all of the qPCR assays were established in a previous study (27). The marker copy number per 100 ml of water was calculated for all samples subjected to qPCR with a cycle threshold (C T ) value above background, and all data were log 10 transformed before statistical analysis.…”

“…We theorize that in order to better assess the impact of fecal pollution it is important to target active cells via rRNA-based detection of gene markers in fecal source tracking studies. Since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells (26), it is not a surprise that recent studies have shown that rRNA-targeted RT-qPCR assays can be more sensitive than rRNA gene-based qPCR assays (27,28). In addition, the rRNA/rRNA gene ratio from each target bacterium is an important indicator of its metabolic status within bacterial communities and has been used to provide an estimate of in situ bacterial growth rates (29,30).…”