Molecular assays (i.e., PCR and qPCR) used in microbial water quality studies often target ribosomal RNA genes (rDNA). However, using DNA as the PCR template does not discriminate between active and dead cells. The use of RNA-based detection methods has recently been proposed as RNA is rather unstable outside of the cell and degrade much faster than DNA in nonactive/dead cells. RNA studies can also take advantage of the higher number of rRNA copies in physiologically active cells. The objective of this study is to discuss some of the latest results on the use of RNA-based methods as an alternate approach for the detection of active bacterial members of a microbial community. Specifically, we have compared fecal bacteria detection rates using rRNA- vs rDNA-based qPCR assays, and evaluated the use of rRNA/rDNA ratios to access the metabolic status of different fecal bacterial groups. We demonstrated that the use of rRNA-based RT-qPCR assays increased the detection frequency of some fecal bacteria and host-specific markers in surface water samples from an urban watershed impacted by combined sewer overflows. Overall, our data show that in many cases using RNA approaches increased microbial detection of fecal bacteria in environmental waters while also providing quantitative information on the active bacterial fraction. However, future epidemiological studies must be conducted to determine whether RNA-based detection increases the correlation between the detection and activity levels of fecal indicators and illness associated with exposure to waterborne pathogens. Moreover, since MST methods may improve risk assessments of different pollution sources, it remains to be seen whether host-specific signals based on metabolically active cells add value to predictive risk models. In spite of these research gaps, our data suggest that RNA-based assays may be used in several applications, including in studies measuring the environmental fate and transport of fecal bacteria.<br/>

Enhanced detection of bacteria in environmental waters: An RNA-based approach

251st National Meeting (2016)

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